Methods for sequencing samples

ABSTRACT

Personalized medicine involves the use of a patient&#39;s molecular markers to guide treatment regimens for the patient. The scientific literature provides multiple examples of correlations between drug treatment efficacy and the presence or absence of molecular markers in a patient sample. Methods are provided herein that permit efficient dissemination of scientific findings regarding treatment efficacy and molecular markers found in patient tumors to health care providers.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.15/183,655, filed Jun. 15, 2016, which is a divisional of U.S.application Ser. No. 14/927,254, filed Oct. 29, 2015, which is acontinuation of U.S. application Ser. No. 14/075,996, filed Nov. 8,2013, which is a continuation of U.S. application Ser. No. 13/060,425,filed Jun. 2, 2011, now U.S. Pat. No. 8,583,380, which is a NationalStage Entry of International Application Serial No. PCT/US2009/056101,filed Sep. 4, 2009, which claims benefit of U.S. Provisional PatentApplication Ser. No. 61/094,855, filed Sep. 5, 2008, U.S. ProvisionalPatent Application Ser. No. 61/155,477, filed Feb. 25, 2009, U.S.Provisional Patent Application Ser. No. 61/173,179, filed Apr. 27, 2009,and U.S. Provisional Patent Application Ser. No. 61/231,287, filed Aug.4, 2009, which applications are incorporated herein by reference intheir entireties.

BACKGROUND OF THE INVENTION

Approximately 50% of Americans will get cancer and approximately 50% ofthose will die from cancer. Ten cancers made up approximately 70% of the1.4 million estimated new U.S. cases in 2008. The NIH estimated that thetotal costs of cancer in 2007 were $219.2 billion, with direct medicalcosts at $89.0 billion (total of all health expenditures), indirectmorbidity costs at $18.2 billion (cost of lost productivity due toillness), and indirect mortality costs at $112.0 billion (cost of lostproductivity due to premature death).

There are approximately 150 oncology drugs, and over 2000 indevelopment. Many drugs have known or measurable targets. However,cancer patients are not routinely screened for the status of thesetargets (or markers), and markers are not used to determine which drugtreatment options the patients receive. Rather, cancers are oftentreated by organ. Furthermore, an integrated source of knowledgeregarding molecular markers and drug targets for physicians or patientsis lacking.

Personalized medicine involves the use of molecular markers thatcharacterize a patient's disease to direct the medical care the patientreceives. The scientific literature provides examples where the efficacyof a cancer drug can be correlated with the status of a molecularmarker. There is a need to improve the ability of health care providersto access and interpret information in the scientific literatureregarding connections between molecular markers and drug efficacy. Thisinformation will allow health care providers to use molecular markers toselect appropriate standard chemotherapy.

Another issue surrounding cancer treatment is the reimbursement of feesassociated with treatments. Medicare can reimburse for off-label use ofcancer drugs included in certain medical compendia. Additionally,peer-reviewed medical journals can provide guidance to Medicarecontractors with respect to medically appropriate off-label uses ofcancer drugs supported by results of clinical trials. There is a need toensure the latest peer-reviewed information regarding off-label use ofcancer drugs and information about experimental drugs supported byresults of clinical trials is readily accessible to physicians andpatients when a cancer drug treatment decision is made or whenreimbursement from an insurance company is being sought.

SUMMARY OF THE INVENTION

In general, in one aspect, a method for generating a personalized cancerdrug treatment option report is provided including obtaining a samplefrom a subject, determining the status of one or more molecular markersin the sample, stratifying one or more cancer drug treatment options inthe report based on the status of the one or more molecular markers, andannotating the report based on the status of the one or more molecularmarkers.

The subject can be a cancer patient. The cancer patient can be a coloncancer patient. The sample can be a tumor biopsy. The sample can bepreserved by formalin-fixing and paraffin-embedding. The determining caninclude nucleic acid amplification, DNA sequencing, FISH, qPCR, and/orIHC. The DNA sequencing can include de novo DNA sequencing. The de novoDNA sequencing can include bridge amplification of DNA. The sample caninclude genomic DNA from an FFPE sample and the determining can includebridge amplification and DNA sequencing.

The method can further include purifying cancer cells from the sample.The purifying can include flow sorting or laser capture microdissection.The sample can be a tumor biopsy obtained by fine needle aspiration, thesample can be frozen, and the purifying can include flow sorting.

The stratifying can include stratifying the drug treatment options for acondition in the NCCN Clinical Practice Guidelines in Oncology™ or theAmerican Society of Clinical Oncology (ASCO) clinical practiceguidelines. The condition can be colon cancer. The stratifying can bebased on information in scientific literature. The stratifying can takeinto account the status of one or more molecular markers in drugabsorption, distribution, metabolism, or excretion genes in a samplefrom the subject. The determining can include analyzing the sequence ofUGT1A1 for one or more SNPs. The stratifying can take into accountwhether the subject is hypermetabolic. The stratifying can take intoaccount the CYP450 status of the subject. The stratifying and/or theannotating can be based on clinical information for the subject. Thestratifying can include ranking drug treatment options with a higherlikelihood of efficacy higher than drug treatment options with a lowerlikelihood of efficacy or for which no information exists with regard totreating subjects with the determined status of the one or moremolecular markers. The stratifying can include indicating on the reportone or more drug treatment options for which scientific informationsuggests the one or more drug treatment options will be efficacious in asubject, based on the status of one or more molecular markers in thesample from the subject. The stratifying can include indicating on areport one or more drug treatment options for which some scientificinformation suggests the one or more drug treatment options will beefficacious in the subject, and some scientific information suggests theone or more drug treatment options will not be efficacious in thesubject, based on the status of one or more molecular markers in thesample from the subject. The stratifying can include indicating on areport one or more drug treatment options for which scientificinformation indicates the one or more drug treatment options will not beefficacious for the subject, based on the status of one or moremolecular markers in the sample from the subject. The stratifying caninclude color coding the listed drug treatment options on the reportbased on the rank of the predicted efficacy of the drug treatmentoptions.

The annotating can include annotating a report for a condition in theNCCN Clinical Practice Guidelines in Oncology™ or the American Societyof Clinical Oncology (ASCO) clinical practice guidelines. The annotatingcan include listing one or more FDA-approved drugs for off-label use,one or more drugs listed in a Centers for Medicare and Medicaid Services(CMS) anti-cancer treatment compendia, and/or one or more experimentaldrugs found in scientific literature, in the report. The annotating caninclude connecting a listed drug treatment option to a referencecontaining scientific information regarding the drug treatment option.The scientific information can be from a peer-reviewed article from amedical journal. The annotating can include using information providedby Ingenuity® Systems. The annotating can include providing a link toinformation on a clinical trial for a drug treatment option in thereport. The annotating can include presenting information in a pop-upbox or fly-over box near provided drug treatment options in anelectronic based report. The annotating can include adding informationto a report selected from the group consisting of one or more drugtreatment options, scientific information concerning one or more drugtreatment options, one or more links to scientific information regardingone or more drug treatment options, one or more links to citations forscientific information regarding one or more drug treatment options, andclinical trial information regarding one or more drug treatment options.

The sample can include colon cancer cells, the one or more molecularmarkers can include c-kit, and the one or more drug treatment optionscan include imatinib mesylate.

The sample can include colon cancer cells, the one or more molecularmarkers can include Kras, and the one or more drug treatment options caninclude cetuximab, panitumumab, or bevacizumab. If exon 2 of Kras doesnot have a mutation, the stratifying and/or annotating can includerecommending cetuximab or panitumumab monotherapy for the subject. Ifexon 2 of Kras has a mutation, the stratifying and/or annotating caninclude recommending cetuximab or panitumumab monotherapy not be givento the subject.

The sample can include colon cancer cells, the one or more molecularmarkers can include BRAF, and the one or more drug treatment options caninclude cetuximab or panitumumab. The determining can includedetermining the presence or absence of the BRAF V600E mutation. If BRAFsequence encodes a V600E mutation, the stratifying and/or annotating caninclude providing a recommendation in the report to the treat thesubject with sorafenib and cetuximab and/or panitumumab.

The determining can include determining the status of microsatellitesequences. If the microsatellite sequences display low-frequencymicrosatellite instability, and the sample is a stage II or stage IIIcolon cancer, the stratifying and/or annotating can include recommendingfluorouracil-based adjuvant chemotherapy for the subject. If themicrosatellite sequences display high-frequency microsatelliteinstability, and the sample is a stage II or stage III colon cancer, thestratifying and/or annotating can include making a recommendation to notadminister fluorouracil-based adjuvant chemotherapy for the subject.

The determining can include determining EGFR copy number. If EGFR copynumber is increased relative to normal, the stratifying and/orannotating can include recommending cetuximab or panitumumab monotherapyto the subject.

The determining can include determining if the sample has 18q chromosomeloss. If the subject is a stage II colorectal cancer patient withchromosome 18q allelic loss the stratifying and/or annotating caninclude recommending adjuvant therapy for the subject.

The determining can include determining thymidylate synthase levels. Ifthymidylate synthase levels are high, the stratifying and/or annotatingcan include recommending adjuvant 5-FU-based chemotherapy not be givento the subject in the report. If thymidylate synthase levels are low,the stratifying and/or annotating can include recommending to treat thesubject with adjuvant 5-FU-based chemotherapy.

The sample can include colon cancer cells, the one or more molecularmarkers can include Topol, and the one or more drug treatment optionscan include 5-FU, irinotecan, or capecitabine. If Topol expression islow, the stratifying and/or annotating can include making arecommendation to not treat the subject with irinotecan. If Topolexpression is moderate to high, the stratifying and/or annotating caninclude making a recommendation to treat the subject with irinotecan.

The method can further include sending a kit to a health care providerthat provides health care to the subject. The kit can include RNAlater®.

The method can further include establishing an in vitro culture usingthe sample. The method can further include high-throughput screening ofFDA approved off-label drugs or experimental drugs using the in vitroculture.

The method can further include establishing a xenograft model using thesample. The method can further include high-throughput screening of FDAapproved off-label drugs or experimental drugs using the xenograftmodel. The sample can be a tumor biopsy, the subject can be a cancerpatient with end stage cancer, and results of the high-throughputscreening can be used to determine an adjuvant therapy foradministration to the cancer patient.

The method can further include monitoring tumor antigen for recurrencedetection.

The determining can include de novo DNA sequencing and the stratifyingcan be based on analysis of a Kaplan-Meier survival curve.

The method can further include charging a fee for generating the report.

In another aspect, a method for generating a personalized cancer drugtreatment option report is provided including: a) obtaining a samplefrom a subject; b) determining the status of one or more molecularmarkers in the sample; c) stratifying drug treatment options listed in aclinical practice guideline for a condition based on the status of theone or more molecular markers in the sample; and d) optionallyannotating the report with information comprising information regardingone or more additional drug treatment options not listed in the clinicalpractice guideline for the condition, wherein the information isincluded based on the status of the one or more molecular markers in thesample. The clinical practice guideline can be the NCCN ClinicalPractice Guidelines in Oncology or the American Society of ClinicalOncology (ASCO) clinical practice guidelines. The one or more additionaldrug treatment options can be described in National Comprehensive CancerNetwork (NCCN) Drugs and Biologics Compendium™, Thomson MicromedexDrugDex®, Elsevier Gold Standard's Clinical Pharmacology, or AmericanHospital Formulary Service—Drug Information Compendium. The one or moreadditional drug treatment options can include a drug used in a clinicaltrial. The one or more additional drug treatment options can include adrug described in a scientific journal article.

The annotating can further include listing information in the reportreferencing one or more scientific journal articles that describe theuse of one or more additional drug treatment options.

The one or more additional drug treatment options can target a molecularmarker that is in a pathway for which the status of another molecularmarker indicates that targeting the pathway would be efficacious fortreating the subject. The stratifying can be indicated by color codingdrug treatment options listed on the report. The color code can includea shade of green, a shade of yellow, and a shade of red. The drugtreatment options associated with a green shade can be drugs that arerecommended to the subject based on the status of the one or moremolecular markers. The drug treatment options associated with a yellowshade can be drugs that have some information that supports recommendingthe drug as a treatment option to the subject based on the status of theone or more molecular markers and at least one piece of information doesnot support recommending the drug as a treatment option. The drugtreatment options associated with a red shade can be drugs for which thestatus of the one or more molecular markers does not supportrecommending the drug treatment option to the subject.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a flow diagram of a personalized medicine service.

FIG. 2A-2C illustrates a physician accessing a report provided by apersonalized medicine business based on the NCCN Clinical PracticeGuidelines in Oncology™ on a computer and finding the drug treatmentoptions for colon cancer stratified based on the status of a molecularmarker present in the cancer.

FIG. 3 illustrates a method for accessing a report provided by apersonalized medicine business based on the NCCN Clinical PracticeGuidelines in Oncology™ on a computer, selecting an annotated drugtreatment, accessing scientific information regarding the drugtreatment, and submitting the information to an insurance company.

FIG. 4 illustrates a flow diagram for a personalized medicine servicefor identifying (determining the status of) molecular markers from asubject and reporting drug treatment options to the subject.

FIG. 5 is a flow diagram for providing personalized treatment options toa patient or physician.

FIG. 6 shows molecular markers or drug targets that can be identified ina subject and drug treatment options that can be recommended to thesubject with the molecular marker, references, and methods of testingthe status of a molecular marker.

FIG. 7 shows an algorithm for determining which drug treatment optionsto recommend to a subject with one or more molecular markers of aparticular status.

FIG. 8 illustrates an example of a report generated by a method of theprovided invention.

FIG. 9 illustrates a process for sequencing DNA from FFPE samples.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

In general, methods are disclosed herein for a service that providespersonalized cancer drug treatment options based on the status of one ormore molecular markers in a patient's sample, e.g., a tumor biopsy. Thedisclosed methods can include stratifying cancer drug treatment optionsand/or annotating cancer drug treatment options with scientificinformation on a report, e.g., a printed form. The scientificinformation can be made accessible to a patient or one or more healthcare providers and can be used for insurance reimbursement purposes toaid selection of one or more drug treatment options.

FIG. 1 provides an overview of one aspect of the provided method. Acancer patient (100) visits a health care provider, and the health careprovider performs a tumor biopsy (102) and potentially begins a firstline drug treatment (104). The tumor biopsy is then sent to apersonalized medicine business for analysis and testing. In one pathway(106), the tumor is used to establish an in vitro culture and/orxenograft model for future drug testing. In another pathway, the tumorcells can be isolated from non-tumor cells in the biopsy (108) and usedto identify (determine the status of) molecular markers (110). Forexample, the status of one or more molecular markers can be determinedby comparative genomic hybridization (CGH), high density expression, denovo sequencing, or genotyping. The molecular markers can then be usedto stratify a list of cancer drug treatments (112). The identifiedmolecular markers can also be used as the basis for annotating the drugtreatment options by, for example, providing links to scientificinformation for experimental drugs and FDA-approved drugs for off-labeluse (114). The links to scientific literature can be used to sendliterature to insurance companies for reimbursement purposes. Thestratified and annotated cancer drug treatment options can then beprovided in the form of a report to the health care provider and/orpatient (116), who can use the information to select a second line drugtreatment therapy, for example, if the first line therapy fails. Thestratified and annotated drug treatment options can be tested inhigh-throughput screens using the established in vitro cultures orxenograft models (118). Results of the screens can be given to thephysician (120), who can use the information to select adjuncts for endstage administration, for example. One or more fees can be charged bythe personalized medicine business in exchange for any of the services.

In one aspect, a method is provided that includes a) obtaining a samplefrom a subject, b) identifying (determining the status of) one or moremolecular markers in the sample, c) stratifying one or more drugtreatment options based on the status of one or more molecular markers,and d) annotating drug treatment options based on the status of one ormore molecular markers.

In another aspect, a method is provided that provides personalizedcancer drug treatment options, including a) receiving an order, b)obtaining a sample from a subject, c) purifying cancer cells from thesample, d) identifying (determining the status of) one or more molecularmarkers in the cancer cells, e) stratifying one or more drug treatmentoptions in a report, f) annotating one or more drug treatment options ina report, and g) charging a fee for one or more of steps (a)-(f).

In another aspect, a method for generating a personalized cancer drugtreatment option report is provided including a) obtaining a sample froma subject; b) determining the status of one or more molecular markers inthe sample; c) stratifying drug treatment options listed in a clinicalpractice guideline for a condition based on the status of the one ormore molecular markers in the sample; and d) optionally annotating thereport with information comprising information regarding one or moreadditional drug treatment options not listed in the clinical practiceguideline for the condition, wherein the information is included basedon the status of the one or more molecular markers in the sample.

Another aspect of a method of the provided invention is shown in FIG. 5,in which a patient or physician interfaces with a personalized medicinebusiness (500), a biopsy is performed (502), a molecular analysis isperformed (504), and information is analyzed to provide recommendationsto the patient or physician (506) based on the status of one or moremolecular markers. The information can be scientific literature and/orclinical trial information. The analysis can include annotating aclinical practice guideline for a condition (e.g., NCCN ClinicalPractice Guidelines in Oncology) with information (e.g., references toscientific journal articles; other drug treatment options). The analysiscan include analysis of molecular pathways that include drug targets,e.g., drug targets not recommended in the clinical practice guidelinefor the condition, that may be efficacious for the patient based on thestatus of one or more molecular markers in the biopsy.

II. Obtaining a Sample

The subject from whom a sample is taken can be a patient, for example, acancer patient or a patient suspected of having cancer. The subject canbe a mammal, e.g., a human, and can be male or female. The sample can bea tumor biopsy. The biopsy can be performed by, for example, a healthcare provider, including a physician, physician assistant, nurse,veterinarian, dentist, chiropractor, paramedic, dermatologist,oncologist, gastroenterologist, or surgeon.

A. Receiving an Order

The methods of the provided invention can include a step in which apersonalized medicine business receives an order. The order received bythe personalized medicine business can be placed by a health careprovider, including, for example, a physician, physician assistant,nurse, veterinarian, dentist, chiropractor, paramedic, dermatologist,oncologist, gastroenterologist, or surgeon. The order can be placed onbehalf of a subject from whom a sample is taken, e.g., a cancer patient.The order can be placed by the subject from whom a sample has been orwill be taken, and the subject can place the order with the guidance orsupervision of a health care provider. The order can be submitted to thepersonalized medicine business using any of a variety of means. Forexample, an order can be placed using a computer website (a webinterface), by sending an email, a facsimile, or a text message, or byplacing a phone call to the personalized medicine business. If a websiteor web interface is used, the website or web interface can be maintainedby the personalized medicine business. The person placing the order cancreate or be given a username and/or password. An order form can bedownloaded from a website, sent through the mail, sent using email, orsent by facsimile. The order can be placed by filling out one or moreforms and submitting it (them) to the personalized medicine businessusing a computer (e.g., web interface, email) or through a mail or otherdelivery service, for example, the U.S. Postal Service, a courierservice, Federal-Express, DHL, or UPS.

A diagnostic biopsy of a primary tumor can be taken before or after anorder is placed. The status of one or more molecular markers in a tumorof the subject can be identified (determined) before or after an orderis placed. The subject from whom the sample is taken can be provided oneor more therapies before or after an order is placed.

The order can include information regarding a sample or subject. Theinformation can include a description of the sample, e.g., the date thesample was taken, type of sample, or other properties of the sample. Theorder can include information regarding the subject from whom the sampleis taken. For example, this information can include height, weight, eyecolor, hair color, age, ethnicity, gender; clinical information, e.g.,blood pressure, LDL cholesterol levels, HDL cholesterol levels, andtriglyceride levels, heart rate; personal medical history, includingcancer treatments already received; family medical history of thesubject; and information on molecular markers.

The order can include billing information and/or insurance information.The order can contain a barcode that identifies the order. The order cancontain comments or notes about the sample and/or the subject (e.g.,patient). This information can be sent with the sample to thepersonalized medicine business.

A customer service representative employed by the personalized medicinebusiness can process the order (e.g., enter information in the orderinto a database operated by the personalized medicine business). Anemployee of the personalized medicine business, for example, a researchnurse, can review and process the clinical information. A fee can becharged by the personalized medicine business in exchange for receivingand/or processing the clinical information.

B. Kit for Processing Samples and/or Submitting Samples to aPersonalized Medicine Business

The personalized medicine business can provide a kit to a health careprovider (e.g., a surgeon, pathologist, oncologist) involved in the careof a subject from whom a sample will be taken. A kit can containcontents that can be used to process or transport a sample from thesubject.

A kit can include a reagent that protects RNA in fresh specimensincluding, e.g., RNAlater® Tissue Collection: RNA Stabilization Solution(Applied Biosystems) or RNASafer™ RNA Stabilization Reagent(SABiosciences). Compositions for preserving RNA in cells and tissuesare described, for example, in U.S. Pat. Nos. 6,204,375, 7,056,673, and7,138,226. A kit of the provided invention can contain instruments ortools for processing a sample.

A kit can contain instructions for processing a sample. The kit cancontain instructions for performing a biopsy. For example, the kit cancontain instructions for performing fine needle aspiration. The kit caninclude instructions and/or reagents for preparing formalin-fixed,paraffin-embedded tissue or for freezing (e.g., fresh frozen) a sample.The kit can contain dry ice (e.g., pelleted dry ice). A kit can containone or more pre-paid shipping labels. A kit can contain educationalmaterials (e.g., a pamphlet containing information about a specificcancer).

A kit of the provided invention can contain instructions for shipping asample, for example, to an entity that will analyze the sample.

A kit can include one or more containers, e.g., an envelope, a box,and/or a tube that can be used to transport one or more samples to thepersonalized medicine business. The one or more containers can beprovided with identifying information, e.g., information identifying thesubject from whom a sample is taken, information identifying a healthcare provider that will perform/has performed a biopsy, informationidentifying the sample type to be sent to the personalized medicinebusiness, and information regarding a shipping address to which thesample can be sent (e.g., address of the personalized medicinebusiness). A kit can contain logistics information; for example, the kitcan contain preprinted information that can be attached to the one ormore sample containers that can be returned to the personalized medicinebusiness. The shipping information can contain a barcode for trackingthe shipment or for identifying the sample. The preprinted informationcan contain an adhesive that permits the information to adhere to acontainer.

One or more logistics and operation personnel employed by thepersonalized medicine business can send a kit to and/or receive a kitfrom a health care provider. A fee can be charged by the personalizedmedicine business in exchange for sending a kit. Multiple individualscan be informed when a kit has been sent by the personalized medicinebusiness; for example, the subject from whom the sample is taken (e.g.,cancer patient) or an oncologist that will review the subject'sinformation, can be informed by the personalized medicine business thata kit has been sent to a health care provider.

A kit can be sent to a pathologist and/or pathology laboratory forpreparing a diagnostic paraffin primary biopsy, and the subject (e.g.,cancer patient) and an oncologist can be notified that the kit has beensent to the pathologist. The pathology laboratory can be a College ofAmerican Pathologists (CAP) accredited laboratory. The laboratory can bea Clinical Laboratory Improvement Amendments (CLIA) certifiedlaboratory. A kit for processing a biopsy can be sent to the person whowill perform the biopsy (e.g., surgeon), and the subject (e.g., patient)and an oncologist can be notified that the kit was sent. A kit can besent to a person (e.g., a surgeon), for example, after surgery, forprocessing a second biopsy.

C. Performing a Biopsy and Submitting a Sample

Any biopsy technique used by those skilled in the art can be used forisolating a sample from a subject. The biopsy can be an open biopsy, inwhich general anesthesia is used or a closed biopsy, in which a smallercut is made than in an open biopsy. The biopsy can be a core orincisional biopsy, in which part of the tissue is removed; an excisionalbiopsy, in which attempts to remove an entire lesion are made; or aneedle aspiration (percutaneous) biopsy (fine needle aspiration biopsy),in which a sample of tissue or fluid is removed with a needle. Theneedle can be a thin, hollow needle, and it can be inserted into a massto extract cells from the mass. Tissue can be obtained by lumpectomy ormastectomy. Tissue can be obtained by colectomy (e.g., a total colectomyor partial colectomy). Tissue can be obtained by a prostatectomy.

The biopsy can be performed by a health care provider, including, forexample, a physician, physician assistant, nurse, veterinarian, dentist,chiropractor, paramedic, dermatologist, oncologist, gastroenterologist,or surgeon.

The biopsy can be processed. For example, the biopsy can be preserved byformalin-fixing and paraffin-embedding (FFPE) the tissue. The biopsy canbe processed into smaller pieces. The biopsy can be treated to preserveRNA, e.g., with RNAlater® or RNASafer™. The biopsy can be stored on wetice (approximately 4° C.), at room temperature (approximately 25° C.),stored at approximately −20° C., or at approximately −80° C., e.g.,stored on dry ice, or frozen in liquid nitrogen or a dry ice/alcoholslurry. The tissue can be frozen within 0.5, 1, 5, 10, 15, 30, 60, 120,or 240 minutes of surgical resection. Fixative agents that can be usedon the biopsy tissue include, for example, methanol-acetone, Carnoy'sfixative (60% ethanol, 30% chloroform, 10% glacial acetic acid), Bouin'sfixative, ethanol, acetone, formalin, methacarn (substitute 60% methanolfor the ethanol in Carnoy), UMFIX (universal molecular fixative),Omnifix, and FINEfix.

The sample can be one or more cells or tissue from, e.g., liver, lung,colon, pancreas, bladder, brain, breast, cervix, esophagus, eye,gallbladder, kidney, stomach, ovary, penis, prostate, pituitary,salivary gland, skin, testicle, uterus, and vagina. The sample can be,for example, blood or urine. The sample can contain nucleic acid, forexample, genomic DNA, amplified genomic DNA, cDNA, amplified cDNA, RNA,amplified RNA, unspliced RNA, spliced RNA, messenger RNA, or microRNA.The sample can be a cell-free sample that contains nucleic acid, forexample, DNA or RNA.

The sample can be a bone marrow core or clot. The clot can bedecalcified. The sample can be one or more unstained slides from an FFPEblock. The one or more slides can be air-dried (unbaked). The slides canbe charged or uncharged. The sample can be a pleural/ascitic fluid. Thepleural/ascitic fluid can be formalin fixed and parafilm-embedded in acell block.

The sample can contain greater than 10% tumor, greater than 15% tumor,greater than 20% tumor, greater than 25% tumor, greater than 30% tumor,greater than 35% tumor, greater than 40% tumor, greater than 45% tumor,greater than 50% tumor, greater than 55% tumor, greater than 60% tumor,greater than 65% tumor, greater than 70% tumor, greater than 75% tumor,greater than 80% tumor, greater than 85% tumor, greater than 90% tumor,greater than 95% tumor, or greater than 99% tumor. The sample cancontain 100% tumor. The type of sample submitted to a personalizedmedicine business can depend on the type of molecular analysis to beperformed on the sample (e.g., DNA microarray, DNA sequencing, IHC).

The sample (e.g., biopsy) can be obtained by a personalized medicinebusiness by receiving it from, for example, the health care provider whoremoved the sample from the subject. The sample can be obtained by apersonalized medicine business through a mail or delivery service; forexample, a physician can perform a tumor biopsy and arrange to send thetumor biopsy. Parafilm-embedded tissue can be sent to a personalizedmedicine business in an envelope. The paraffin embedded sample can be adiagnostic primary biopsy. A fresh-frozen sample can be sent to apersonalized medicine business in a container, and the container caninclude dry ice. A blood sample can be sent in a container, e.g., atube. A sample can be sent in a container with an ice-water mixture. Asample can be sent to a personalized medicine business in a containerprovided by the personalized medicine business.

One or more employees of a personalized medicine business can review thesamples that are obtained by the personalized medicine business. Thesamples can be reviewed by a pathologist employed by the personalizedmedicine business. The personalized medicine business can request thatone or more additional samples be sent to the personalized medicinebusiness, for example, after receiving an initial sample from thesubject.

D. Purifying Cancer Cells

The methods of the provided invention can include a step in whichprimary tumor cells are separated/purified from non-tumor cells in asample, e.g., a biopsy sample. Any technique used by those skilled inthe art can be appropriate for isolating primary tumor cells fromnon-tumor cells in the tumor biopsy. For example, the tumor cells can beisolated using techniques that include optical cell sorting,flow-cytometry, flow-sorting, fluorescence activated cell sorting(FACS), magnetic cell sorting (MACS; e.g., using antibody coatedmagnetic particles), size-based separation (e.g., using a sieve, anarray of obstacles, a filter), sorting in a microfluidics device,antibody-based separation, sedimentation, affinity adsorption, affinityextraction, or density gradient centrifugation. Fresh-frozen or FFPEsamples can be used for flow-sorting. Sorting can be based on cell size,morphology, or intracellular or extracellular markers. Methods forisolating or sorting tumor cells are described, for example, in NagrathS. et al. (2007) Nature 450:1235-1239; U.S. Pat. Nos. 6,008,002,7,232,653, and 7,332,288; PCT Publication No. WO2008157220A1; and USPatent Application Nos. US20080138805A1 and US20090186065; and RosenbergR. et al. (2002) Cytometry 49:150-158, each of which is hereinincorporated by reference in their entireties.

Fluorescence-activated cell sorting (FACS) uses light scattering andfluorescent characteristics to sort cells. A fluorescent property can beimparted on a cell using, e.g., nucleic acid probes or antibodiesconjugated to a fluorescent dye. A cell suspension can form a stream offlowing liquid. The stream of cells forms drops that containapproximately one cell per drop. Before the stream forms drops, afluorescent characteristic of each cell is measured. A charge is placedon an electrical charging ring prior to fluorescence intensitymeasurement and the opposite charge is carried on the drop as it breaksfrom the stream. The charged drops pass through two high voltagedeflection plates that divert drops into different containers based upontheir charge. The charge can be directly applied to the stream and thedrop breaking off retains the charge of the same sign as the stream. Thestream is then returned to neutral after the drop breaks off.

Direct or indirect immunofluorescence can be used in FACS. In directimmunofluorescence, an antibody is directly conjugated to a fluorescentdye. In indirect immunofluorescence, the primary antibody is notlabeled, and a secondary antibody is conjugated to a fluorescent dye.

Different types of cancer cells can be sorted based on one or moredifferent molecular markers, and these markers can be on the surface ofthe cancer cells. Cells, e.g., circulating tumor cells, can be isolatedfrom a sample, e.g., blood, using technology from, e.g., CELLective DxCorporation.

A sample can be obtained from a subject, e.g., a colon cancer patient,by fine needle aspiration biopsy, the sample can be stored fresh-frozen,and the cancer cells in the sample can be isolated by flow-sorting.

Cancer cells can be purified by laser capture microdissection. In lasercapture microdissection, a transparent transfer film is applied on thesurface of a tissue section. Cells to be removed are identified using amicroscope, and a near infrared (IR) laser diode is activated. The laserbeam fuses the transfer film to the underlying cells. The film can thenbe removed with the desired cells attached. Fresh-frozen and paraffinembedded tissue may be used for laser capture microdissection.

Cancer cells can be purified using Panomics (Affymetrix®) Cancer CellIsolation Kit (Catalog number CI0002, CI0004, or CI0010). Cancer cellscan be isolated using CytoSelect™ Clonogenic Tumor Cell Isolation Kit(Catalog number CBA-155 or CBA-155-5) from Cell Biolabs, Inc.

A fee can be charged by a personalized medicine business in exchange forpurifying cancer cells from a sample.

III. Molecular Markers

A. Techniques for Determining the Status of Molecular Markers

The methods of the provided invention can include analyzing a sample forthe status of one or more molecular markers. The sample can be analyzedfor one or more molecular markers that can include, for example, nucleicacids, including DNA and RNA, proteins, including antibodies,autoantibodies, and cell surface receptors, gene or protein expressionprofiles, carbohydrates, and lipids. The status of one or more molecularmarkers in the sample can be identified (determined) using techniquesthat include, for example, comparative genomic hybridization (CGH) orchromosomal microarray analysis (CMA), in which copy number changes ofDNA in a tumor are detected, expression profiling, DNA microarray,high-density oligonucleotide microarray, whole-genome RNA expressionarray, peptide microarray, enzyme-linked immunosorbent assay (ELISA),genome sequencing, de novo sequencing, 454 sequencing, pyrosequencing,Helicos True Single Molecule Sequencing, SOLiD™ sequencing, SOLEXAsequencing, nanosequencing, chemical-sensitive field effect transistor(chemFET) array sequencing, polony sequencing, copy number variation(CNV) analysis sequencing, small nucleotide polymorphism (SNP) analysis,immunohistochemistry (IHC), immunoctyochemistry (ICC), massspectrometry, tandem mass spectrometry, matrix-assisted laser desorptionionization time of flight mass spectrometry (MALDI-TOF MS), in-situhybridization, fluorescent in-situ hybridization (FISH), chromogenicin-situ hybridization (CISH), silver in situ hybridization (SISH),polymerase chain reaction (PCR), digital PCR (dPCR), reversetranscription PCR, quantitative PCR (Q-PCR), single marker qPCR,real-time PCR, nCounter Analysis (Nanostring technology), Westernblotting, Southern blotting, SDS-PAGE, gel electrophoresis, and Northernblotting.

The status of one or more molecular markers corresponding to geneticpolymorphisms between members of a population can be detected bynumerous methods well-established in the art e.g., PCR-based sequencespecific amplification, restriction fragment length polymorphisms(RFLPs), isozyme markers, northern analysis, allele specifichybridization (ASH), array based hybridization, amplified variablesequences of the genome, self-sustained sequence replication, simplesequence repeat (SSR), single nucleotide polymorphism (SNP), randomamplified polymorphic DNA (“RAPD”) or amplified fragment lengthpolymorphisms (AFLP).

Techniques for determining the status of one or more molecular markerscan use hybridization of a probe nucleic acid to nucleic acidscorresponding to the molecular marker (e.g., amplified nucleic acidsproduced using genomic DNA as a template). Hybridization formats forallele detection can include, for example, solution phase, solid phase,mixed phase, or in situ hybridization assays. A guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes Elsevier, N.Y.

The status of a molecular marker can include, for example, geneexpression level (e.g., messenger RNA level) or protein expressionlevel. The expression level can be, for example, higher than normal,normal, or below normal. The status of a molecular marker can includeabsence of a mutation (e.g., wild-type) or presence of one or moremutations (e.g., de novo mutation, nonsense mutation, missense mutation,silent mutation, frameshift mutation, insertion, substitution, pointmutation, single nucleotide polymorphism (SNP), deletion, rearrangement,amplification, chromosomal translocation, interstitial deletion,chromosomal inversion, loss of heterozygosity, loss of function, gain offunction, dominant negative, or lethal); nucleic acid modification(e.g., methylation); or presence or absence of a post-translationalmodification on a protein (e.g., acetylation, alkylation, amidation,biotinylation, glutamylation, glycosylation, glycation, glycylation,hydroxylation, iodination, isoprenylation, lipoylation, prenylation,myristoylation, farnesylation, geranylgeranylation, ADP-ribosylation,oxdiation, palmitoylation, pegylation, phosphatidylinositol addition,phosphopantetheinylation, phosphorylation, polysialyation, pyroglutamateformation, arginylation, sulfation, or selenoylation).

B. De Novo DNA Sequencing Techniques

The methods of the provided invention can use de novo sequencing todetermine the status of one or more molecular markers in a sample, e.g.,a sample from a colon cancer patient. De novo sequencing techniquesinclude, for example, Helicos True Single Molecule Sequencing (tSMS)(Harris T. D. et al. (2008) Science 320:106-109). In the tSMS technique,a DNA sample is cleaved into strands of approximately 100 to 200nucleotides, and a polyA sequence is added to the 3′ end of each DNAstrand. Each strand is labeled by the addition of a fluorescentlylabeled adenosine nucleotide. The DNA strands are then hybridized to aflow cell, which contains millions of oligo-T capture sites that areimmobilized to flow cell surface. The templates can be at a density ofabout 100 million templates/cm². The flow cell is then loaded into aninstrument, e.g., HeliScope™ sequencer, and a laser illuminates thesurface of the flow cell, revealing the position of each template. A CCDcamera can map the position of the templates on the flow cell surface.The template fluorescent label is then cleaved and washed away. Thesequencing reaction begins by introducing a DNA polymerase and afluorescently labeled nucleotide. The oligo-T nucleic acid serves as aprimer. The polymerase incorporates the labeled nucleotides to theprimer in a template directed manner. The polymerase and unincorporatednucleotides are removed. The templates that have directed incorporationof the fluorescently labeled nucleotide are detected by imaging the flowcell surface. After imaging, a cleavage step removes the fluorescentlabel, and the process is repeated with other fluorescently labelednucleotides until the desired read length is achieved. Sequenceinformation is collected with each nucleotide addition step.

Another example of a de novo DNA sequencing technique that can be usedto determine the status of one or more molecular markers in a sample is454 sequencing (Roche) (Margulies, M et al. 2005, Nature, 437, 376-380).454 sequencing involves two steps. In the first step, DNA is shearedinto fragments of approximately 300-800 base pairs, and the fragmentsare blunt ended. Oligonucleotide adaptors are then ligated to the endsof the fragments. The adaptors serve as primers for amplification andsequencing of the fragments. The fragments can be attached to DNAcapture beads, e.g., streptavidin-coated beads using, e.g., Adaptor B,which contains 5′-biotin tag. The fragments attached to the beads arePCR amplified within droplets of an oil-water emulsion. The result ismultiple copies of clonally amplified DNA fragments on each bead. In thesecond step, the beads are captured in wells (pico-liter sized).Pyrosequencing is performed on each DNA fragment in parallel. Additionof one or more nucleotides generates a light signal that is recorded bya CCD camera in a sequencing instrument. The signal strength isproportional to the number of nucleotides incorporated.

Pyrosequencing makes use of pyrophosphate (PPi) which is released uponnucleotide addition. PPi is converted to ATP by ATP sulfurylase in thepresence of adenosine 5′ phosphosulfate. Luciferase uses ATP to convertluciferin to oxyluciferin, and this reaction generates light that isdetected and analyzed.

Another example of a de novo DNA sequencing technique that can be usedto determine the status of one or more molecular markers in a sample isSOLiD technology (Applied Biosystems). In SOLiD sequencing, genomic DNAis sheared into fragments, and adaptors are attached to the 5′ and 3′ends of the fragments to generate a fragment library. Alternatively,internal adaptors can be introduced by ligating adaptors to the 5′ and3′ ends of the fragments, circularizing the fragments, digesting thecircularized fragment to generate an internal adaptor, and attachingadaptors to the 5′ and 3′ ends of the resulting fragments to generate amate-paired library. Next, clonal bead populations are prepared inmicroreactors containing beads, primers, template, and PCR components.Following PCR, the templates are denatured and beads are enriched toseparate the beads with extended templates. Templates on the selectedbeads are subjected to a 3′ modification that permits bonding to a glassslide.

The sequence can be determined by sequential hybridization and ligationof partially random oligonucleotides with a central determined base (orpair of bases) that is identified by a specific fluorophore. After acolor is recorded, the ligated oligonucleotide is cleaved and removedand the process is then repeated.

Another example of a de novo sequencing technology that can be used todetermine the status of one or more molecular markers in a sample isSOLEXA sequencing (Illumina). SOLEXA sequencing is based on theamplification of DNA on a solid surface using fold-back PCR and anchoredprimers. Genomic DNA is fragmented, and adapters are added to the 5′ and3′ ends of the fragments. DNA fragments that are attached to the surfaceof flow cell channels are extended and bridge amplified. The fragmentsbecome double stranded, and the double stranded molecules are denatured.Multiple cycles of the solid-phase amplification followed bydenaturation can create several million clusters of approximately 1,000copies of single-stranded DNA molecules of the same template in eachchannel of the flow cell. Primers, DNA polymerase and fourfluorophore-labeled, reversibly terminating nucleotides are used toperform sequential sequencing. After nucleotide incorporation, a laseris used to excite the fluorophores, and an image is captured and theidentity of the first base is recorded. The 3′ terminators andfluorophores from each incorporated base are removed and theincorporation, detection and identification steps are repeated.

Another example of de novo sequencing technology that can be used todetermine the status of one or more molecular markers in a sample is thesingle molecule, real-time (SMRT™) technology of Pacific Biosciences. InSMRT, each of the four DNA bases is attached to one of four differentfluorescent dyes. These dyes are phospholinked. A single DNA polymeraseis immobilized with a single molecule of template single stranded DNA atthe bottom of a zero-mode waveguide (ZMW). A ZMW is a confinementstructure which enables observation of incorporation of a singlenucleotide by DNA polymerase against the background of fluorescentnucleotides that rapidly diffuse in an out of the ZMW (in microseconds).It takes several milliseconds to incorporate a nucleotide into a growingstrand. During this time, the fluorescent label is excited and producesa fluorescent signal, and the fluorescent tag is cleaved off. Detectionof the corresponding fluorescence of the dye indicates which base wasincorporated. The process is repeated.

Another example of de novo sequencing that can be used to determine thestatus of one or more molecular markers in a sample is nanoporesequencing (Soni G V and Meller A. (2007) Clin Chem 53: 1996-2001). Ananopore is a small hole, of the order of 1 nanometer in diameter.Immersion of a nanopore in a conducting fluid and application of apotential across it results in a slight electrical current due toconduction of ions through the nanopore. The amount of current whichflows is sensitive to the size of the nanopore. As a DNA molecule passesthrough a nanopore, each nucleotide on the DNA molecule obstructs thenanopore to a different degree. Thus, the change in the current passingthrough the nanopore as the DNA molecule passes through the nanoporerepresents a reading of the DNA sequence.

Another example of de novo sequencing that can be used to determine thestatus of one or more molecular markers in a sample involves using achemical-sensitive field effect transistor (chemFET) array to sequenceDNA (as described in US Patent Application Publication No. 20090026082).In one example of the technique, DNA molecules can be placed intoreaction chambers, and the template molecules can be hybridized to asequencing primer bound to a polymerase. Incorporation of one or moretriphosphates into a new nucleic acid strand at the 3′ end of thesequencing primer can be detected by a change in current by a chemFET.An array can have mulitiple chemFET sensors. In another example, singlenucleic acids can be attached to beads, and the nucleic acids can beamplified on the bead, and the individual beads can be transferred toindividual reaction chambers on a chemFET array, with each chamberhaving a chemFET sensor, and the nucleic acids can be sequenced.

Any one of the de novo sequencing techniques described herein can beused to determine the status of one or more molecular markers in themethods of the provided invention.

DNA can be extracted from an FFPE sample and the DNA can be bridgeamplified and sequenced. The sequences generated can be used todetermine the status (e.g., wild-type or mutant) of one or moremolecular markers (e.g., genes). An example of the process isillustrated in FIG. 9. Greater than 1 μg of DNA can be extracted (902)from an FFPE sample (900). Adaptor sequences can be ligated to the DNAin an in vitro ligation reaction (904). The extracted DNA can be bridgeamplified to generate a library of single molecules that are spatiallyclustered (906). The bridge amplified DNA can then be sequenced byserial extension of primed templates (908). The accuracy of sequencereads can be up to 35 bp (910). The sequences can be used to determinethe status (e.g., wild-type or mutant) of one or more genes in thesample.

DNA from an FFPE tissue sample, including a historic FFPE tissue sample,can be sequenced, for example using 454 sequencing, to determine thestatus of one or more molecular markers (Thomas R K et al. (2006) NatureMedicine 12:852-855). Sequences from one or more historic samples can becompared to sequences from a sample from a subject, e.g., a cancerpatient. The status of one or more molecular markers in a historicsample can be correlated with one or more treatment outcomes, and thecorrelation of a treatment outcome with molecular marker status in oneor more historic samples can be used to predict treatment outcomes forthe subject, e.g., a cancer patient. These predictions can be the basisfor determining whether or not to recommend a drug treatment option tothe subject.

The status of one or more molecular markers can be determined by de novoDNA sequencing of samples from cancer patients, and cancer patients canbe grouped based on the status of the one or more molecular markers. Forinstance, cancer patients with wild-type Kras can be grouped together,and patients with mutant Kras can be grouped together. The survival ofuntreated patients in each group can be followed over time, and patientsurvival can be plotted to generate a Kaplan-Meier curve. A Kaplan-Meierplot can be used to illustrate the fraction of patients alive after acertain amount of time. The survival of cancer patients in each groupafter treatment with a drug can be followed over time, and patientsurvival can be plotted to generate a Kaplan-Meier curve. Analysis of apreviously generated Kaplan-Meier curve can be used to recommend one ormore drug treatment options to a subject (e.g., a cancer patient), basedon the status of one or more molecular markers in a sample from thesubject (e.g., a cancer patient).

C. Business Considerations

A personalized medicine business can outsource or sub-contract thedetermining of the status of one or more molecular markers to a ClinicalLaboratory Improvement Amendments (CLIA) certified laboratory. Thepersonalized medicine business can outsource or sub-contract thedetermining of the status of one or more molecular markers, for example,whole genome analysis, to another company, for example, Illumia® orAffymetrix®. Mass spectrometry (e.g., matrix-assisted laser desorptionionization time of flight mass spectrometry (MALDI-TOF MS)) can beperformed at, e.g., Biodesix. Only a portion of the whole genomeanalysis data can be reported to a patient and/or health care provider,and the rest of the data can be stored by the personalized medicinebusiness. The stored data can be sent to the subject and/or health careprovider at a later date.

Other in vitro or in vivo tests can be performed on a sample. Thepersonalized medicine business can outsource or sub-contract anothercompany to perform the in vitro or in vivo tests.

A fee can be charged by the personalized medicine business in exchangefor performing the techniques and/or identifying (determining the statusof) one or more molecular markers. The molecular marker analysis can beperformed by one or more employees of the personalized medicinebusiness, for example, one or more laboratory technicians.

IV. Drug Metabolism

The methods of the provided invention can include analyzing a samplefrom a subject for the status of one more markers related to drugabsorption, distribution, metabolism, and excretion. The sample to beanalyzed can be, for example, a tumor biopsy, blood, cheek (buccal)swab, or other fluid or tissue taken from the subject. Any test known bythose skilled in the art to investigate drug absorption, distribution,metabolism, or excretion genes can be used. For example, the Affymetrix®Drug Metabolizing Enzymes and Transporters (DMET)—Early Access Solutioncan be used to analyze drug metabolism biomarkers. CYP450 status can beassessed using the Roche® AmpliChip CYP450 Test, which tests for genevariations in CYP2D6 and CYP2C19. The Human1M BeadChip from Illumina®,Human1M-Duo DNA Analysis BeadChip from Illumina®, and HumanExon510S-DuoDNA Analysis BeadChip from Illumina® can be used to identify SNPs inADME (absorption, distribution, metabolism, and excretion) genes. Thestatus of ADME associated genes can be investigated using DTEx™ GeneExpression Analysis from NoAb BioDiscoveries.

A sample can be analyzed for one or more SNPs in the UGT1A1 gene, whichencodes UDP-glucuronosyltransferase. Identification of UGT1A1 promoterpolymorphisms can be used to predict the toxicity of treatment withCPT-111 (irinotecan). Irinotecan is a topoisomerase I inhibitor that canbe used for treating colon cancer. Irinotecan is activated by hydrolysisto SN-38, and SN-38 is inactivated by glucuronidation by UGT1A1.Subjects with the *28 variant ((TA)₇ allele) of UGT1A1 express reducedamounts of UGT1A1 in their liver, and they do not clear irinotecan asrapidly as others. This can correspond to increased rates of severediarrhea and neutropenia. Patients with polymorphisms in the UGT1A1 gene(e.g., *28 variant) have been recommended by the FDA to receive reduceddoses of irinotecan. If a sample from a subject, e.g., a cancer patient,has a *28 variant UGT1A1 allele, a recommendation can be made in areport to treat the subject with a reduced amount of irinotecan.

A fee can be charged by the personalized medicine business for analyzingone or more molecular markers related to absorption, distribution,metabolism, or excretion of one or more drugs. The analysis of one ormore molecular markers related to drug absorption, distribution,metabolism, or excretion can be performed by one or more laboratorytechnicians employed by the personalized medicine business.

Stratifying drug treatment options and annotating drug treatment optionsin a report regarding the use of a drug treatment option can be donebased on the status of one or more molecular markers that regulate drugabsorption, distribution, metabolism, or excretion in a sample from asubject.

V. Stratifying Drug Treatment Options

The methods of the provided invention can include using the status ofone or more molecular markers identified in a sample to stratify (rank)drug treatment options for the subject from whom the sample was taken(target-drug approach). The stratifying of drug treatments can be basedon scientific information regarding the molecular markers. For example,the scientific information can be data from one or more studiespublished in one or more scientific journals (e.g., New England Journalof Medicine (NEJM), Lancet, etc.). The scientific information can bedata provided in a commercial database (e.g., data stored in a databaseprovided by Ingenuity® Systems). One or more pieces of scientificinformation can be used to stratify the treatments.

A. Classes of Drugs

Drug treatment options can be stratified into classes based on thestatus of one or more molecular markers in a sample. For example, afirst class of drug treatment options can be those for which scientificinformation predicts a drug will be efficacious for a subject whosesample has one or more molecular markers of a particular status. Drugsin this first class can be a recommended drug treatment option for asubject.

A second class of drug treatment options can be those for which somescientific information predicts a drug will be efficacious for a subjectwith one or more molecular markers of a particular status, and somescientific information does not support use of the drug for the subject,based on one or more molecular markers of a particular status in asample from a subject. For example, a sample may contain a marker whosestatus indicates the drug will be efficacious in the subject and anothermarker (e.g., a mutant version of a drug metabolism gene) that indicatesthe drug would also have a toxic affect on the subject. Up to 9molecular markers in a sample from a patient can indicate that a drug islikely to be efficacious for treating a subject from whom the sample wastaken, and one molecular marker, for instance a drug metabolism marker,can indicate that the drug would have a high level of toxicity in thesubject.

This second class can also include drugs for which there is indirectscientific support for drug efficacy in a subject (e.g., the drugtargets a protein that is in the same molecular pathway as a molecularmarker in a sample). For example, a drug in this class could target akinase that functions downstream of an overexpressed cell surfacereceptor that is known to be targeted by an efficacious drug. A drug inthis second class can be a recommended drug treatment option for asubject.

A third class of drugs can be those for which scientific informationindicates the drug will not be efficacious in the subject based on thestatus of one or more molecular markers in a sample from the subject.For example, a drug that targets a cell surface receptor may not displayefficacy if a downstream kinase is mutated. It can be recommended that asubject not be treated with a drug in this third class.

The drug treatment options can be stratified using an algorithm-basedapproach. An example of an algorithm for selecting a marker is shown inFIG. 7. The status of one or more molecular markers in a patient sampleis determined (700). The scientific literature is analyzed forinformation related to the status of the molecular marker and theefficacy of one or more different drugs (702, 704). If the status of amolecular marker correlates with efficacy of a drug, then arecommendation can be made to treat the subject with that drug (706). Ifthe status of a molecular marker does not correlate with efficacy of adrug, then a recommendation can be made not to treat a subject with thedrug (708). A computer and computer readable medium can be used tostratify the drug treatment options.

A list of stratified drug treatment options can be presented in the formof a report. The stratification of drug treatment options can beindicated by color coding. For example, drugs in the first class can becolor coded in green, drugs in the second class can be color coded inyellow, and drugs in the third class can be color coded in red.

The recommendation of a drug treatment option for a subject can be basedon the stage of the cancer of the subject, e.g., a late stage cancer.Drug treatment options can also be stratified based on other factors,e.g., the type of cancer, age of the subject, status of drug metabolismgenes (genes involved in absorption, distribution, metabolism, andexcretion), efficacy of other drugs the patient has received, clinicalinformation regarding the subject, and family medical history.

A fee can be charged by the personalized medicine business in exchangefor stratifying the drug treatment options. The drug treatment optionscan be stratified by one or more employees of the personalized medicinebusiness, for example, a molecular pathologist and research nurse.

B. Examples of Molecular Marker/Drug Relationships and Drugs Used forCancer Treatment

Drug treatment options can be stratified based on a relationship betweenthe status of one or more molecular markers in the sample and theefficacy of a drug treatment used on other subjects with the one or moremolecular markers of a particular status. The scientific literaturecontains multiple examples of drugs that have efficacy in treatingsubjects with tumors with certain molecular markers.

The status of c-kit (also known as CD117, KIT, PBT, SCFR) in a samplefrom a subject can be determined by, e.g., expression or DNA sequencing,and can be used to determine whether to recommend imatinib mesylate(Gleevec) as a drug treatment option for the subject, e.g., agastrointestinal tumor patient with a metastatic and/or unresectablemalignant tumor (NCCN Clinical Practice Guidelines in Oncology; HenrichM C et al. (2003) J. Clin. Oncol. 21:4342-4349). C-kit is a receptor forcytokine stem cell factor (SCF, steel factor, or c-kit ligand). Whenc-kit binds to SCF it forms a dimer which activates signaling throughsecond messengers. Signaling through c-kit plays a role in cellsurvival, proliferation, and differentiation. Imatinib mesylate(Gleevec) is an inhibitor of receptor tyrosine kinases. If a sample froma subject with a metastatic and/or unresectable malignantgastrointestinal stromal tumor expresses c-kit or c-kit with activatingmutations, a recommendation can be made to treat the subject withimatinib mesylate (Gleevec). If a sample from a subject does not expressc-kit or c-kit with activating mutations, a recommendation can be madeto not treat the subject with imatinib mesylate (Gleevec). Imatinibmesylate (Gleevec) is efficacious in GIST patients with activatingmutations in the juxtamembrane (JM) domain of c-kit.

The presence of Bcr-Abl in a sample from a subject can be used todetermine whether to recommend imatinib mesylate (Gleevec) as a drugtreatment option to the subject, e.g., a chronic myeloid leukemia (CML)patient. For example, a patient with CML with the Philadelphiachromosome (Philadelphia translocation) that produces the Bcr-Ablprotein, a tyrosine kinase, can be recommended to be treated withimatinib mesylate (Gleevec). Approximately 95% of people with CML havethe Philadelphia chromosome. Imatinib mesylate (Gleevec) can berecommended as an initial therapy for a CML patient. Imatinib mesylate(Gleevec) can be recommended for use while a CML patient is in the firstphase of CML (the chronic phase) (Kantarjian H. et al. (2002) NEJM356:645-652). Imatinib mesylate (Gleevec) can be recommended to a CMLpatient if interferon alpha therapy fails (Druker B J et al. (2001)NEJM344:1031-1037).

The status of PDGFR in a sample can be used to determine whether torecommend imatinib mesylate (Gleevec) as a drug treatment option to asubject, e.g., a cancer patient. Cancer cells overexpressing plateletderived growth factor receptor (PDGFR), a tyrosine kinase, or withactivating mutations in PDGFR can be targeted with imatinib mesylate(Gleevec) (Heinrich M C et al. (2003) J. Clin. Oncol. 21:4342-4349). Ifa sample from a subject overexpresses PDGFR, or expresses PDGFR withactivating mutations, a recommendation can be made to treat the subjectwith imatinib mesylate (Gleevec). If a sample from a subject does notoverexpress PDGFR, a recommendation can be made to not treat the subjectwith imatinib mesylate (Gleevec).

A subject with renal cell carinoma (RCC) or imatinib-resistant GIST thatexpresses the marker PDGFR can be treated with Sutent (Sunitib orSU11248), a receptor tyrosine kinase inhibitor. If a sample from asubject with RCC or imatinib-resistant GIST expresses PDGFR, arecommendation can be made to treat the subject with Sutent (Sunitib).

The status of secreted protein acidic and rich in cysteine (SPARC; alsoknown as ON, osteonectin) in a sample can be used to determine whetherto recommend Abraxane as a drug treatment option for a subject, e.g., ametastatic breast cancer patient. SPARC is a matrix-associated proteinthat can elicit alterations in cell shape, inhibit cell-cycleprogression, and influence the synthesis of the extracellular matrix(ECM). Pre-clinical studies indicate that a patient with a tumorexpressing SPARC, which binds albumin, can be targeted with a paclitaxelalbumin-stabilized nanoparticle formulation (Abraxane) and that Abranxemay have a higher level of efficacy in these patients compared withpatients whose tumors do not express SPARC. If a sample from a subjectexpresses SPARC, a recommendation can be made to treat the subject withAbraxane. If a sample from a subject does not express SPARC, arecommendation can be made to not treat the subject with Abraxane.Abraxane is indicated for the treatment of breast cancer after failureof combination chemotherapy for metastatic disease or relapse within 6months of adjuvant chemotherapy.

The status of HSP90 (also known as HSPN; LAP2; HSP86; HSPC1; HSPCA;Hsp89; HSP89A; HSP90A; HSP90N; HSPCAL1; HSPCAL4; FLJ31884; HSP90AA1) ina sample can be used to determine whether to recommend CNF2024 (BIIB021)as a drug treatment option for a subject. Hsp90 is a molecular chaperonethat can facilitate the maturation and stabilization of mutated,overexpressed and constitutively or persistently active oncoproteins(Taldone T, Curr Opin Pharmacol. (2008) August; 8(4):370-374). A patientwith GIST expressing heat shock protein 90 (HSP90) can be targeted withCNF2024 (BIIB021), an oral inhibitor of HSP90. If a sample from asubject expresses HSP90, a recommendation can be made to treat thesubject with CNF2024 (BIIB021). If a sample from a subject does notexpress HSP90, a recommendation can be made to not treat the subjectwith CNF2024 (BIIB021).

The status of MGMT (O-6-methylguanine-DNA methyltransferase) promotermethylation in a sample can be used to determine whether to recommendtemozolomide (Temodar, Temodal) as a drug treatment option for asubject, e.g., a glioblastoma patient. The MGMT gene is located onchromosome 10q26. MGMT encodes a DNA-repair protein that removes alkylgroups from the 06 position of guanine, a site of DNA alkylation.06-methylguanine lesions induced by chemotherapy that remain unrepairedcan trigger cytotoxicity and apoptosis. High levels of MGMT activity incancer cells can reduce the therapeutic effect of an alkylating agent.Loss of MGMT expression and reduced DNA-repair activity are associatedwith epigenetic silencing of the MGMT gene by promoter methylation.Temozolomide (Temodar, Temodal) is an alkylating agent. Data suggestthat MGMT promoter methylation is associated with a favorable outcomeafter temozolomide chemotherapy in patients with recently diagnosedglioblastoma (Hegi M E et al. (2005) NEJM 352:997-1003). If a samplefrom a subject has MGMT promoter methylation, a recommendation can bemade to treat the subject with temozolomide. If a sample from a subjectdoes not have MGMT promoter methylation, a recommendation can be made tonot treat the subject with temozolomide.

The status of HER2 (also known as ERBB2, NEU, NGL, TKR1, CD340, HER-2,HER-2/neu) in a sample can be determined by, e.g., IHC, PCR,SISH/CISH/FISH, and can be used to determine whether to recommendtrastuzumab (Herceptin) as a drug treatment option for a subject, e.g.,a breast cancer patient. HER2 is an epidermal growth factor receptor.HER2 can bind to other ligand-bound EGF receptor family members to forma heterodimer. This binding can stabilize ligand binding and enhancekinase-mediated activation of downstream signaling pathways, such asthose involving mitogen-activated protein kinase andphosphatidylinositol-3 kinase. Amplification and/or overexpression ofHER2 is reported in numerous cancers, including breast and ovariantumors. Breast cancer patients that overexpress the HER2 receptor can betreated with trastuzumab (Herceptin), a monoclonal antibody that canbind to the domain IV of the extracellular segment of HER2. Herceptin isapproved for adjuvant treatment of HER2-overexpressing metastatic breastcancer. As a single agent, Herceptin is approved for treatment ofHER2-overexpressing breast cancer in patients who have received one ormore chemotherapy regimens for metastatic disease. If a sample from asubject (e.g., a breast cancer patient) overexpresses HER2, arecommendation can be made to treat the subject with trastuzumab(Herceptin). If a sample from a subject (e.g., a breast cancer patient)does not overexpress HER2, a recommendation can be made to not treat thesubject with trastuzumab (Herceptin).

The status of human epidermal growth factor receptor 1 (also known asHER1, EGFR, ERBB, mENA, ERBB1, PIG61) in a sample can be used todetermine whether to recommend Erlotinib (Tarceva) as a drug treatmentoption for a subject. EGFR is a transmembrane glycoprotein receptor forepidermal growth factor family members. Binding of the EGFR to a ligandinduces receptor dimerization and tyrosine autophosphorylation and leadsto cell proliferation. Mutations in HER1 are associated with lungcancer. Erlotinib (Tarceva) binds to the ATP binding site of the EGFRtyrosine kinase. Tarceva monotherapy is indicated for the treatment ofpatients with locally advanced or metastatic non-small cell lung cancerafter failure of at least one prior chemotherapy regimen. Tarceva incombination with gemcitabine is indicated for the first-line treatmentof patients with locally advanced, unresectable or metastatic pancreaticcancer. Erlotinib (Tarceva) is efficacious in patients with pancreaticcancer and non-small cell lung cancer that are positive byimmunohistochemistry (IHC) for EGFR. If a sample from a subjectexpresses EGFR, erlotinib (Tarceva) can be recommended as a drugtreatment option for the subject. If a sample from a subject does notexpress EGFR, a recommendation can be made to not treat the subject witherlotinib (Tarceva).

The status of vascular endothelial growth factor (VEGF) can be used todetermine whether to recommend Bevacizumab (Avastin) as a drug treatmentoption. VEGF is a glycosylated mitogen that can act on endothelialcells. VEGF can mediate increased vascular permeability, inducingangiogenesis, vasculogenesis and endothelial cell growth, promote cellmigration, and inhibit apoptosis. Bevacizumab (Avastin) is a recombinanthumanized monoclonal antibody directed to VEGF. Bevacizumab (Avastin)can block tumor growth by inhibiting the formation of new blood vessels.Bevacizumab (Avastin) is indicated for first or second-line treatment ofpatients with metastatic carcinoma of the colon or rectum in combinationwith intravenous 5-fluorouracil-based chemotherapy. Bevacizumab(Avastin) is also indicated for the first-line treatment ofunresectable, locally advanced, recurrent or metastatic non-squamousnon-small cell lung cancer in combination with carboplatin andpaclitaxel. Bevacizumab (Avastin) is also indicated for the treatment ofpatients who have not received chemotherapy for metastatic HER2-negativebreast cancer in combination with paclitaxel. Bevacizumab (Avastin) isindicated for the treatment of metastatic renal cell carcinoma incombination with interferon alpha. If a sample from a subject expressesVEGF, Bevacizumab (Avastin) can be recommended as a drug treatmentoption. If a sample from a subject does not express VEGF, arecommendation can be made to not treat the subject with Bevacizumab(Avastin).

The status of ER (also known as estrogen receptor; ESR; Era; ESRA;NR3A1; DKFZp686N23123; ESR1) in a sample from a subject can bedetermined by, e.g., IHC, and can be used to determine whether torecommend hormonal therapeutics to the subject, e.g., a subject withbreast cancer. ER is a ligand-activated transcription factor thatincludes domains for hormone binding, DNA binding, and activation oftranscription. ER can localize to the nucleus and can form a homodimeror a heterodimer with estrogen receptor 2. If a sample, e.g., a biopsyfrom a breast cancer patient, is analyzed by IHC and receives a scorethat establishes it as ER-positive, a recommendation can be made totreat the subject with hormonal therapeutics. If the sample from asubject (e.g., a breast cancer patient) is determined to be ER-negative,a recommendation can be made to not treat the subject with hormonaltherapeutics.

The status of PR (also known as progesterone receptor; NR3C3; PGR) in asample from a subject can be determined by, e.g., IHC, and can be usedto determine whether to recommend hormonal therapeutics to the subject,e.g., a subject with breast cancer. PR is a member of the steroidreceptor superfamily and mediates the physiological effects ofprogesterone. If a sample, e.g., a biopsy from a breast cancer patient,is analyzed by IHC and receives a score that establishes it asPR-positive, a recommendation can be made to treat the subject withhormonal therapeutics. If the sample from a subject (e.g., a breastcancer patient) is determined to be PR-negative (by, e.g., IHC), arecommendation can be made to not treat the subject with hormonaltherapeutics.

The expression status of 21 genes in a sample from a subject (e.g., abreast cancer patient) can be determined using the Oncotype Dx assay(Genomic Health Inc.), which involves reverse transcription followed byRT-PCR of FFPE samples from primary breast cancer. The levels ofexpression are used to calculate an RS (Recurrence Score®), which can beused to assign a patient to a treatment group. The score generated fromthe analysis can be used to determine whether to recommend tamoxifen asa drug treatment option for a breast cancer patient. Tamoxifen is aselective estrogen receptor modulator (SERM). The Oncotype Dx test canbe used after surgery (e.g., lumpectomy or mastectomy) but before adecision on adjuvant therapy is made.

The status of the PAM 50 expression signature in sample from a subjectcan be determined by, e.g., RT-PCR, and can be used to determine whetherto recommend endocrine therapy for the subject, e.g., a breast cancerpatient (Parker J S et al. (2009) J Clin Oncol. 27:1160-1167). The50-gene PAM50 subtype predictor can be used to assign intrinsic subtypesto tumor samples and can be used to assess the likelihood of efficacyfrom neoadjuvant chemotherapy.

The status of vras and Kras in a sample from a subject can be used todetermine whether to recommend bevacizumab (Avastin) as a drug treatmentoption to the subject, e.g., a colon cancer patient or a patient withmetastatic colon cancer. (Grothey A and Turja J H (2008)cme.medscape.com/viewarticle/577606).

Capecitabine (Xeloda) can be recommended as an adjuvant to a subjectwith colorectal cancer Stage III (Dukes' C) colon cancer (Twelves C. etal. (2005) NEJM 352:2696-2704) or as a first-line monotherapy formetastatic colorectal cancer. Capecitabine (Xeloda) is a prodrug thatcan be converted to 5-fluorouracil (5-FU) in a tumor. 5-FU inhibits DNAsynthesis, thereby slowing tumor growth.

The status of a VeriStrat® (Biodesix) serum protein signature in asample from a subject can be determined by, e.g., matrix-assisted laserdesorption ionization time of flight mass spectrometry (MALDI-TOF MS),and can be used to determine whether to recommend cetuximab (Erbitux) asa drug treatment option for the subject, e.g., a colon cancer patient.The VeriStrat® test can predict the response to EGFR tyrosine kinaseinhibitors (e.g., gefitinib or erlotinib) for patients with non-smallcell lung cancer (NSCLC), and can be used to determine whether torecommend erlobtinib (Tarceva) or gefitinib (Iressa) to a subject, e.g.,a metastatic lung cancer patient, non-small-cell lung cancer patient,adenocarinoma (ADC) patient, or squamous cell carcinoma (SCC) patient.Erlotinib (Tarceva) can target the epidermal growth factor receptor(EGFR) tyrosine kinase.

The status of EGFR expression in a sample from a subject can be used todetermine whether to recommend cetuximab (Erbitux) as a drug treatmentoption for a subject, e.g., a colon cancer patient with an irinotecanresistant tumor (irinotecan (Camptosar) is a topoisomerase 1 inhibitor).For example, if a sample from a subject is determined to be anEGFR-expressing, metastatic colorectal carcinoma and the subject isrefractory to irinotecan-based chemotherapy, a recommendation can bemade to treat the subject with cetuximab (Erbitux).

The status of TOPO1 (also known as DNA topoisomerase; TOPI; TOP1)expression in a sample from a subject can be determined by, e.g., IHC,and can be used to determine whether to recommend fluorouracil (5-FU;FSU; Adrucil) with or without irinotecan or oxaliplatin for the subject,e.g., a colon cancer patient (Braun M. S. et al. (2008) J Clin Oncol.26:2690-2698). TOPO1 can alter the topologic states of DNA by catalyzingthe transient breaking and rejoining of a single strand of DNA whichallows the strands to pass through one another. Fluorouracil (Adrucil)is a pyrimidine analog that can act as an inhibitor of thymidylatesynthase, which makes thymidine for DNA replication. If a biopsy from acolon cancer patient is determined to have low TOPO1 expression by,e.g., IHC, 5-FU (Adrucil) can be recommended as a drug treatment optionwithout irinotecan or oxaliplatin. If a biopsy from a colon cancerpatient is determined to have moderate or high TOPO1 expression by,e.g., IHC, 5-FU (Adrucil) treatment with irinotecan or oxaliplatin canbe recommended as a drug treatment option for the colon cancer patient.

The status of TOPO1 in a sample from a subject can be used to determinewhether to recommend irinotecan (Camptosar) as a drug treatment optionfor the subject, e.g., a metastatic colon cancer patient. Irinotecan canbe recommended to a metastatic colon cancer patient after recurrence ofcolon cancer or progression after 5-FU therapy.

The status of Phosphatase and Tensin Homolog (PTEN) in a sample from asubject can be used to determine whether to recommend cetuximab(Erbitux) or panitumumab (Vectibix) for the subject, e.g., a metastaticcolorectal cancer patient (Frattini M. et al. (2007) Br J Cancer97:1139-1145). PTEN can regulate the cell cycle. PTEN dephosphorylatesphosphoinositide substrates. PTEN can regulate the intracellular levelsof phosphatidylinositol-3,4,5-triphosphate and can act as a tumorsuppressor by negatively regulating the Akt/PKB signalling pathway. If asample from a subject expresses PTEN, a recommendation can be made totreat the subject with cetuximab (Erbitux) or panitumumab (Vectibix). Ifa sample from a subject does not express PTEN, a recommendation can bemade to not treat the subject with cetuximab (Erbitux) or panitumumab(Vectibix).

The status of PIK3CA in a sample from a subject can be used to determinewhether to recommend cetuximab (Erbitux) or panitumumab (Vectibix) forthe subject, e.g., a metastatic colorectal cancer patient(Satore-Bianchi A et al. (2009) Cancer Res 69:1851-1857). PIK3CA encodesthe 110 kDa catalytic subunit or phosphatidylinositol 3-kinase (which isalso composed of an 85 kDa regulatory subunit). The catalytic subunitcan phosphorylate PtdIns, PtdIns4P and PtdIns(4,5)P2. Membranelocalization of PIK3CA inhibits PTEN and promotes AKT1 phosphorylation.If a sample from a subject has wild-type PIK3CA, a recommendation can bemade to treat the subject with cetuximab (Erbitux) or panitumumab(Vectibix). If a sample from a subject has mutations in PIK3CA, arecommendation can be made to not treat the subject with cetuximab(Erbitux) or panitumumab (Vectibix).

Leucovorin (Wellcovorin, folinic acid) can be recommended as a drugtreatment option for a subject, e.g., a colon cancer patient, incombination with 5-FU to prolong survival and promote palliative care.Leucovorin enhances the binding of 5-FU to thymidylate synthase and as aresult prolongs the life span of 5-FU. This results in an anti-cancereffect of 5-FU.

Levamisole (Ergamisol) can be recommended as a drug treatment option fora subject, e.g., a patient with Dukes' Stage C colon cancer, incombination with 5-FU after surgical resection.

Oxaliplatin (Eloxatin) can be recommended as a drug treatment option fora subject, e.g., a patient with metastatic colon cancer, in combinationwith 5-FU and leucovorin (LV). Oxaliplatin (Eloxatin) is aplatinum-based chemotherapy drug thought to inhibit DNA synthesis.Oxaliplatin (Eloxatin) used in combination with infusional 5-FU/LV isindicated for adjuvant treatment of stage III colon cancer patients whohave undergone complete resection of the primary tumor and for treatmentof advanced carcinoma of the colon or rectum.

Celecoxib (Celebrex) can be recommended as a drug treatment option for asubject, e.g., a colon cancer patient, a patient with familialadenomatous polyposis (FAP), a patient with colonic polyps, or a patientwith colonic polyposis syndrome (FAPC) (Bertagnolli M M et al. (2006)NEJM 355:873-884; Arber N et al. (2006) NEJM 355:885-895). Celecoxib(Celebrex) is a nonsteroidal anti-inflammatory drug (NSAID) that is aspecific COX-2 inhibitor.

The status of Kras (also known as v-Ki-ras2 Kirsten rat sarcoma viraloncogene homolog; NS3; KRAS1; KRAS2; RASK2; KI-RAS; C—K-RAS; K-RAS2A;K-RAS2B; K-RAS4A; K-RAS4B) in sample from a subject can be used todetermine whether to recommend cetuximab (Erbitux) as a drug treatmentoption to the subject, e.g., a colon or rectal cancer patient with stageIV disease (COL-5, COL-9, COL-10) (NCCN Clinical Practice Guidelines inOncology). Kras is a member of the small GTPase superfamily. Cetuximab(Erbitux), a monoclonal antibody that targets the epidermal growthfactor receptor (EGFR), is efficacious in colorectal cancer patientswith wild-type Kras (Karapetis C S et al. NEJM 359, 1757-1765).Cetuximab is indicated for treatment of locally or regionally advancedsquamous cell carcinoma of the head and neck in combination withradiation therapy and recurrent or metastatic squamous cell carcinoma ofthe head and neck progressing after platinum-based therapy. If a samplefrom a subject has wild-type Kras, a recommendation can be made to treatthe subject with cetuximab (Erbitux). If a sample from a subject hasmutated Kras, a recommendation can be made to not treat the subject withcetuximab (Erbitux). If a sample from a subject has mutations in Kras incodon 12 or 13, a recommendation can be made to not treat the subjectwith cetuximab (Erbitux). A recommendation can be made to treat asubject, e.g., a colon or rectal cancer patient, with cetuximab(Erbitux) if the patient is not able to tolerate cetuximab andirinotecan.

Irinotecan (Camptosar) can be recommended as a drug treatment option fora subject, e.g., a colon or rectal cancer patient (NCCN ClinicalPractice Guidelines in Oncology). Irinotecan (Camptosar) is indicated asa component of first-line therapy in combination with 5-fluorouracil andleucovorin for patients with metastatic carcinoma of the colon orrectum. Irinotecan (Camptosar) is also indicated for patients withmetastatic carcinoma of the colon or rectum whose disease has recurredor progressed following initial fluorouracil-based therapy.

The status of epidermal growth factor receptor (EGFR) can be used todetermine whether to recommend panitumumab (Vectibix) as a drugtreatment option to a subject, e.g., a metastatic colon or rectal cancerpatient. Panitumumab (Vectibix) is a human monoclonal antibody that cantarget epidermal growth factor receptor (EGFR). Panitumumab (Vectibix)is indicated as a single agent for the treatment of EGFR-expressingmetastatic colorectal carcinoma (mCRC) with disease progression on orfollowing fluoropyrimidine-, oxaliplatin-, and irinotecan-containingchemotherapy regimens. If a sample from a subject is determined to be anEGFR-expressing metastatic colorectal cancer, panitumumab (Vectibix) canbe recommended as a drug treatment option for the subject. If a samplefrom a subject is not an EGFR-expressing metastatic colorectal cancer, arecommendation can be made to not treat the subject with panitumumab(Vectibix).

The status of Kras in a sample from a subject can be used to determinewhether to recommend panitumumab (Vectibix) as a drug treatment optionfor the subject, e.g., a metastatic colon or rectal cancer patient withstage IV disease (COL-5, COL-9, COL-10) (NCCN Clinical PracticeGuidelines in Oncology). If a metastatic colorectal cancer sample from asubject has wild-type Kras, a recommendation can be made to treat thesubject with panitumumab (Vectibix). If a metastatic colorectal cancersample from a subject has mutated Kras, a recommendation can be made notto treat the subject with panitumumab (Vectibix). If a metastaticcolorectal cancer sample from a subject has mutations in Kras in codons12 or 13, a recommendation can be made to not treat the subject withpanitumumab (Vectibix). A recommendation can be made to treat a subject,e.g., a colon or rectal cancer patient, with panitumumab (Vectibix) ifthe subject is not able to tolerate cetuximab and irinotecan.

Mitomycin C (Mitozytrex) can be recommended as a drug treatment optionfor a subject, e.g., a gastrointestinal cancer patient, for palliativetreatment of disseminated adenocarcinoma of the stomach or pancreas.Mitomycin C is a DNA crosslinker.

Cyclophosphamide (Neosar, Cytoxan) can be recommended as a drugtreatment option for treating a subject, e.g., a lung cancer patient.Cyclophosphamide is a nitrogen mustard alkylating agent.

The status of Nrf2 (also known as nuclear factor (erythroid-derived2)-like 2; NFE2L2) in a sample from a subject can be used to determinewhether to recommend doxorubicin (Adriamycin) as a drug treatment optionfor the subject, e.g., a lung cancer patient (Wang et al. (2008)Carcinogenesis 29:1235-1243). Overexpression of Nrf2 resulted inenhanced resistance of cancer cells to doxorubicin (Adriamycin). NFE2,NFE2L1, and NFE2L2 comprise a family of human genes encoding basicleucine zipper (bZIP) transcription factors. Doxorubicin (Adriamycin)can intercalate DNA. If a sample from a subject overexpresses Nrf2, arecommendation can be made to not treat the subject with doxorubicin(Adriamycin). If a sample from a subject does not overexpress Nrf2, arecommendation can be made to treat the subject with doxorubicin(Adriamycin).

The status of DPD (also known as dihydropyrimidine dehydrogenase; DHP;DHPDHASE; MGC70799; MGC132008; DPYD) in a sample from a subject can bedetermined by, e.g., tandem mass spectrometry, and can be used todetermine whether to recommend fluorouracil (5-FU) as a drug treatmentoption for the subject, e.g., a lung cancer patient or a non-small-celllung cancer patient (Nakano J et al. (2006) British Journal of Cancer95:607-615). DPD protein is a pyrimidine catabolic enzyme and theinitial and rate-limiting factor in the pathway of uracil and thymidinecatabolism. Mutations in DPD can result in dihydropyrimidinedehydrogenase deficiency, an error in pyrimidine metabolism associatedwith thymine-uraciluria and an increased risk of toxicity in cancerpatients receiving 5-fluorouracil chemotherapy. If a sample from asubject is negative for DPD expression, a recommendation can be made totreat the subject with 5-FU. If a sample from a subject is positive forDPD expression, a recommendation can be made to not treat the subjectwith 5-FU. 5-FU is a pyrimidine analog that can act as a thymidylatesynthase inhibitor. A dose of 5-FU can be recommended based on the levelof DPD expression in a sample from a subject.

The status of OPRT (also known as uridine monophosphate synthetase; UMPSuridine monophosphate synthase; OPRtase; OMPdecase; UMP synthase;orotidine 5′-phosphate decarboxylase; orotate phosphoribosyltransferasephosphoribosyltransferase; orotate phosphoribosyl transferase;orotidine-5′-decarboxylase) in a sample from a subject can be used todetermine whether to recommend 5-FU as a drug treatment option for thesubject, e.g., a lung cancer patient or a non-small-cell lung cancerpatient (Nakano J et al. (2006) British Journal of Cancer 95:607-615).For example, if a sample from a subject is positive for OPRT expression,a recommendation can be made to treat the subject with 5-FU. If a samplefrom a subject is negative for OPRT expression, a recommendation can bemade to not treat the subject with 5-FU.

The status of TS (also known as thymidylate synthetase; TMS; TSase;HsT422; MGC88736; TYMS) in a sample from a subject can be used todetermine whether to recommend 5-FU as a drug treatment option for thesubject, e.g., a lung cancer patient or a non-small-cell lung cancerpatient (Nakano J et al. (2006) British Journal of Cancer 95:607-615;Cascinu S et al. (2001) Ann Oncol 2:239-244). Thymidylate synthasecatalyzes the methylation of deoxyuridylate to deoxythymidylate using5,10-methylenetetrahydrofolate (methylene-THF) as a cofactor. Thisfunction maintains the dTMP (thymidine-5-prime monophosphate) poolcritical for DNA replication and repair. TS is considered to be theprimary site of action for 5-fluorouracil,5-fluoro-2-prime-deoxyuridine, and some folate analogs. If a sample froma subject is negative for TS expression (or has low TS expression), arecommendation can be made to treat the subject with 5-FU. If a samplefrom a subject is positive for TS expression (or has high TSexpression), a recommendation can be made to not treat the subject with5-FU.

Bevacizumab (Avastin) can be recommended as a drug treatment option fora subject, e.g., a lung or NSCLC patient.

Docetaxel (Taxotere) can be recommended as a drug treatment option for asubject, e.g., a metastatic lung cancer patient, a non-small-cell lungcancer patient, a breast cancer patient, a prostate cancer patient, agastric cancer patient, or a head and neck cancer patient. Docetaxel(Taxotere) an anti-mitotic that stabilizes microtubules. Docetaxel(Taxotere) is indicated for the treatment of patients with locallyadvanced or metastatic breast cancer after failure of priorchemotherapy, and it is indicated in combination with doxorubicin andcyclophosphamide for the adjuvant treatment of patients with operablenode-positive breast cancer. Docetaxel (Taxotere) as a single agent isindicated for the treatment of patients with locally advanced ormetastatic non-small cell lung cancer (NSCLC) after failure of priorplatinum-based chemotherapy. Docetaxel (Taxotere) in combination withcisplatin is indicated for the treatment of patients with unresectable,locally advanced or metastatic NSCLC who have not previously receivedchemotherapy for this condition. Docetaxel (Taxotere) in combinationwith prednisone is indicated for the treatment of patients with androgenindependent (hormone refractory) metastatic prostate cancer. Docetaxel(Taxotere) in combination with cisplatin and fluorouracil is indicatedfor the treatment of patients with advanced gastric adenocarcinoma,including adenocarcinoma of the gastroesophageal junction, who have notreceived prior chemotherapy for advanced disease. Docetaxel (Taxotere)in combination with cisplatin and fluorouracil is indicated for theinduction treatment of patients with locally advanced squamous cellcarcinoma of the head and neck (SCCHN).

The status of Kras in a sample from a subject can be used to determinewhether to recommend gefitinib (Iressa) as a drug treatment option forthe subject, e.g., a lung cancer patient or a non-small-cell lung cancerpatient (Carbone D P (2004) Nature Clinical Practice Oncology 1:66-67).For example, if a sample from a subject expresses wild-type Kras, arecommendation can be made to treat the subject with gefitinib (Iressa).If a sample from a subject expresses mutated Kras, a recommendation canbe made to not treat the subject with gefitinib (Iressa). If a samplefrom a subject has mutations in Kras in exon 2, a recommendation can bemade to not treat the subject with gefitinib (Iressa) (Pao W et al.(2005) PLoS Med 2(1): e17. doi:10.1371/journal.pmed.0020017). Gefitinib(Iressa) is an anticancer drug that can inhibit tyrosine kinase activityof EGFR.

Gemcitabine (Gemzar) can be recommended as a drug treatment option for asubject, e.g., a lung cancer patient, a ovarian cancer patient, apancreatic cancer patient, or a NSCLC patient. Gemcitabine (Gemzar) is anucleoside analog in which the hydrogen atoms on the 2′ carbons ofdeoxycytidine are replaced by fluorine atoms. Gemcitabine (Gemzar) canbe recommended as a first line drug treatment option for inoperablestage IIIA, IIIB, or metastatic stage IV lung cancer. Gemcitabine(Gemzar) is indicated in combination with carboplatin for a patient withovarian cancer that has returned at least 6 months after the patient hadfinished platinum-based therapy. Gemcitabine (Gemzar) is indicated incombination with cisplatin for the first-line treatment of patients withlocally advanced (Stage IIIA or Stage IIIB) or metastatic (Stage IV orcancer that has spread) non-small cell lung cancer for whom surgery isnot possible. Gemcitabine (Gemzar) in combination with paclitaxel isapproved by the FDA for the first-line treatment of patients withmetastatic breast cancer after they have received anthracycline, unlesstheir medical condition did not allow them to receive an anthracycline.Gemcitabine (Gemzar) is indicated as a single agent first-line treatmentfor patients with locally advanced (Stage II or Stage III when surgeryis not an option) or metastatic (Stage IV) adenocarcinoma of thepancreas. Gemcitabine (Gemzar) is also indicated for pancreatic cancerpatients previously treated with 5-FU.

Vinorelbine (Navelbine) can be recommended as a drug treatment optionfor a subject, e.g., a lung cancer patient, a breast cancer patient, orNSCLC patient with and unresectable advanced tumor. Vinorelbine(Navelbine) can be recommended as a drug treatment option alone or incombination with cisplatin. Vinorelbine (Navelbine) can be recommendedas a drug treatment option for a patient with Stage III or Stage IV lungcancer or NSCLC. Vinorelbine (Navelbine) is a 5′NOR semi-synthetic vincaalkaloid that can interfere with microtubule assembly.

The status of Kras or EGFR in a sample from a subject can be used todetermine whether to recommend erlotinib (Tarceva) as a drug treatmentoption for the subject, e.g., a lung cancer patient, non-small-cell lungcancer patient, adenocarinoma (ADC) patient, or squamous cell carcinoma(SCC) patient (Pao W. et al. (2005) PLOS Medicine 2(1):e17). For exampleif a sample has wild-type Kras, a recommendation can be made to treatthe subject with erlotinib (Tarceva). If a sample has mutated Kras, arecommendation can be made to not treat the subject with erlotinib(Tarceva). If a sample has mutated EGFR, a recommendation can be made totreat the subject with erlotinib (Tarceva). If a sample from a subjecthas mutations in exons 18-21 of EGFR, a recommendation can be made totreat the subject with erlotinib (Tarceva). If a sample has wild-typeEGFR, a recommendation can be made to not treat the subject witherlotinib (Tarceva). Erlotinib (Tarceva) can be recommended as a firstline therapy.

A recommendation can be made to administer topotecan (Hycamtin) to asubject, e.g., a lung cancer patient or a small cell lung cancerpatient, that has failed a first line of therapy or has cancerprogression after an initial response to chemotherapy (O'Brien M E etal. (2006) J Clin Oncol 24:5441-5447). Topotecan (Hycamtin) is atopoisomerase 1 inhibitor.

Pemetrexed disodium (Alimta) can be recommended as a drug treatmentoption for a subject, e.g., a lung cancer patient or non-small-cell lungcancer patient that has locally advanced or metastatic cancer.Pemetrexed disodium is a member of the folate antimetabolite class ofchemotherapy drugs. Pemetrexed can inhibit three enzymes used in purineand pyrimidine synthesis: thymidylate synthase (TS), dihydrofolatereductase (DHFR), and glycinamide ribonucleotide formyltransferase(GARFT). Pemetrexed for injection (Alimta) is approved by the FDA incombination with cisplatin for the initial treatment of advancednonsquamous non-small cell lung cancer (NSCLC). Pemetrexed for injection(Alimta) as a single agent is approved for maintaining the initialtreatment effect of chemotherapy in patients with advanced nonsquamousnon-small cell lung cancer whose disease has not worsened after initialtherapy. Pemetrexed for injection (Alimta) is approved by the FDA as asingle agent for the treatment of patients with advanced nonsquamousnon-small cell lung cancer (NSCLC) after prior chemotherapy. Pemetrexedfor injection (Alimta) is a treatment for malignant pleural mesothelioma(MPM), a cancer that affects the inside lining of the chest cavity.Pemetrexed for injection (Alimta) can be given with cisplatin whensurgery is not an option.

Porfimer sodium (Photofrin) can be recommended as a drug treatmentoption for a subject, e.g., a lung cancer patient or non-small-cell lungcancer patient who is undergoing photodynamic therapy. Porfimer sodiumis a sensitizer that can be administered intravenously for use inphotodynamic therapy. Photofrin is indicated for palliation of patientswith completely obstructing esophageal cancer, or of patients withpartially obstructing esophageal cancer who, in the opinion of theirphysician, cannot be satisfactorily treated with Nd:YAG laser therapy.Photofrin is indicated for treatment of microinvasive endobronchialnon-small-cell lung cancer (NSCLC) in patients for whom surgery andradiotherapy are not indicated. Photofrin is indicated for reduction ofobstruction and palliation of symptoms in patients with completely orpartially obstructing endobronchial NSCLC.

Paclitaxel (Taxol) can be recommended as a drug treatment option for asubject, e.g., a lung cancer patient or non-small-cell lung cancerpatient, as a first line therapy and/or in combination with cisplatin.Paclitaxel is a mitotic inhibitor that stabilizes microtubules.

Bleomycin (Blenoxane) can be recommended as a drug treatment option fora subject, e.g., a patient with pleural effusion or malignant pleuraleffusion (MPE), the accumulation of fluid in the pleural space that canresult from cancer metastasis. Bleomycin (Blenoxane) can be recommendedto be used in the management of the following conditions as either asingle agent or in combination with other chemotherapeutic agents:squamous cell carcinoma (head and neck, including: mouth, tongue,tonsil, nasopharynx, oropharynx, sinus, palate, lip, buccal mucosa,gingivae, epiglottis, skin, larynx; penis, cervix, and vulva); Hodgkin'sDisease and non-Hodgkin's lymphoma; and testicular carcinoma (embryonalcell, choriocarcinoma, and teratocarcinoma). Bleomycin (Blenoxane) is aglycopeptide that can induce DNA strand breaks.

Leucovorin (folinic acid, Wellcovorin) can be recommended as a drugtreatment option for a subject, e.g., a prostate cancer patient, incombination with 5-FU to prolong survival or provide palliative care.

The status of EGFR can be determined by, e.g., sequencing or detectionof expression, and be used to determine whether to recommend lapatinibas a drug treatment option (Trowe T. et al. (2008) Clinical CancerResearch 14:2465-2475). Lapatinib is a tyrosine kinase inhibitor. If asample overexpresses EGFR, a recommendation can be made to treat thesubject with lapatinib. If a sample does not overexpress EGFR, arecommendation can be made to not treat the subject with lapatinib.

The status of ErbB2 (also known as HER2, NEU, NGL, TKR1, CD340, HER-2,HER-2/neu) can be used to determine whether to recommend leucovorin as adrug treatment option for a subject, e.g, a cancer patient (NCCN).

The status of TOPO1 can be determined by, e.g., IHC, and can be used todetermine whether to recommend oxaliplatin as a drug treatment optionfor a subject, e.g., a colon cancer patient (Braun M. S. et al. (2008) JClin Oncol 26:2690-2698). Oxaliplatin is thought to inhibit DNAsynthesis.

Etoposide phosphate (Etopophos) can be recommended as a drug treatmentoption for a subject, e.g., a lung cancer or small cell lung cancerpatient. Etoposide phosphate is an inhibitor of topoisomerase II.

In the methods of the provided invention, one or more recommendations ina report for treating or not treating a subject with one or more drugscan be made based on one or more correlations between one or moremolecular markers of a particular status and one or more drugs. Arecommendation in a report (e.g., treat with a drug or do not treat witha drug) can be made based on the subject's type of cancer.

C. Examples of Marker/Drug Combinations for Colon Cancer

In the methods of the provided invention a drug can be recommended as atreatment option (first class or second class) or recommended not to beused as a treatment option (third class) based on the status of one ormore molecular markers in a sample in a subject, e.g., a colon cancerpatient.

The status of c-kit (CD117) in a sample from a colon cancer patient canbe determined by, e.g., flow cytometry and/or sequencing, and imatinibmesylate (Gleevec) can be recommended for the subject based on thestatus of c-kit. For example, if flow cytometry indicates the expressionof c-kit in the sample, and the protein sequence has key mutations,imatinib mesylate (Gleevec) can be recommended for the subject as a drugtreatment option. If a sample from a colon cancer patient does notexpress c-kit or have c-kit with key mutations, a recommendation can bemade to not treat the subject with imatinib mesylate (Gleevec).

The status of the Kras in a sample from a subject can be determined by,e.g., sequencing, and cetuximab (Erbitux) or panitumumab (Vectibix)monotherapy can be recommended for the subject based on the status ofthe Kras sequence. For example, if the sample from a colon cancerpatient has a wild type Kras sequence (e.g., as determined by exon 2genotyping or sequencing), cetuximab (Erbitux) or panitumumab (Vectibix)monotherapy can be recommended as a drug treatment option for the coloncancer patient. If the sample from a colon cancer patient has a mutantKras sequence (e.g., as determined by exon 2 genotyping or sequencing),a recommendation can be made not to treat the colon cancer patient withcetuximab (Erbitux) or panitumumab (Vectibix) monotherapy.

The status of BRAF in a sample from a colon cancer patient can bedetermined by, e.g., sequencing, and cetuximab (Erbitux) or panitumumab(Vectibix) can be recommended to a subject based on the status of BRAF(Nicolantonio D et al. (2008) J Clin. Oncol. 26:5705-5712; Cappuzzo F etal. (2008) Br J Cancer 99:83-89). For example, if a subject does nothave a V600E mutation in its BRAF sequence (e.g., as determined by V600Egenotyping or sequencing), cetuximab (Erbitux) or panitumumab (Vectibix)can be recommended as a drug treatment option. If a subject has a V600Emutation in its BRAF sequence (e.g., as determined by V600E genotypingor sequencing), then a recommendation can be made not to treat thepatient with cetuximab (Erbitux) or Panitumumab (Vectibix). Treatment ofsubjects with the BRAF V600E mutation with the BRAF inhibitor sorafenibcan restore sensitivity to cetuximab (Erbitux) or panitumumab (Vectibix)monotherapy. If a sample from a subject has a V600E mutation in its BRAFsequence, then a recommendation can be made to treat the subject withsorafenib and cetuximab (Erbitux) and/or panitumumab (Vectibix).

The presence or absence of microsatellite stability in a sample from asubject, e.g., a colon cancer patient, can be used to determine whetheror not to recommend fluorouracil-based adjuvant chemotherapy to thesubject. Fluorouracil-based adjuvant chemotherapy benefited patientswith stage II or stage III colon cancer with microsatellite-stabletumors or tumors exhibiting low-frequency microsatellite instability butnot those with tumors exhibiting high-frequency microsatelliteinstability (Ribic C M et al. (2003) NEJM 349:247-257). If a sample froma subject with stage II or stage III colon cancer has amicrosatellite-stable tumor or a tumor exhibiting low-frequencymicrosatellite instability, a recommendation can be made to treat thesubject with fluorouracil-based adjuvant chemotherapy. If a subject withstage II or stage III colon cancer has a tumor that exhibitshigh-frequency microsatellite instability, a recommendation can be madenot to treat the subject with fluorouracil-based adjuvant chemotherapy.Microsatellite stability can be determined by DNA sequencing.

The status of EGFR copy number in a sample from a subject, e.g. a coloncancer patient, can be determined by, e.g., FISH or qPCR, and can beused to determine whether to recommend cetuximab (Erbitux) orpanitumumab (Vectibix) to the subject. An increased copy number of EGFRcorrelates with a good response to cetuximab (Erbitux) or panitumumab(Vectibix) monotherapy (Cappuzzo F et al. (2007) Annals of Oncology19:717-723). If a sample from a subject has an increased copy number ofEGFR, a recommendation can be made to treat the subject with cetuximab(Erbitux) or panitumumab (Vectibix) monotherapy.

The status of the 18q chromosome in a sample from a subject, e.g., acolon cancer patient, can be determined by, e.g., qPCR, and can be usedto determine whether to treat the subject with adjuvant therapy (Jen J.et al. (1994) NEJM 331:213-221). The status of chromosome 18q hasprognostic value in patients with stage II colorectal cancer. Stage IIcolorectal cancer patients with chromosome 18q allelic loss have aprognosis that is similar to that in patients with stage III cancer, whoare thought to benefit from adjuvant therapy. In contrast, stage IIcolorectal cancer patients who do not have chromosome 18q allelic lossin their tumor have a survival rate similar to that of patients withstage I disease and may not require additional therapy. If a sample froma subject (e.g., a stage II colon cancer patient) has chromosome 18qallelic loss, then a recommendation can be made to treat the subjectwith adjuvant therapy as if the patient were a stage III cancer patient.If a sample from a subject (e.g., a stage II colon cancer patient) doesnot have chromosome 18q allelic loss, a recommendation can be made tonot treat the subject with adjuvant therapy.

The status of thymidylate synthase levels in a sample from a subject,e.g., a colon cancer patient, can be determined by, e.g., IHC, and canbe used to determine whether to treat the subject with 5-FU-basedchemotherapy (Elder D. (2002) J. Clin. Oncol. 20:1721-1728; Cascinu S etal. (2001) Ann Oncol 2:239-244; Ciaparrone M. et al. (2006) Oncology70:366-377).

If a sample from a subject (e.g., a colon cancer patient) has a high TSlevel (as determined by, e.g., IHC), a recommendation can be made totreat or to not treat the subject with adjuvant 5-FU-based chemotherapy.If a sample from a subject (e.g., a colon cancer patient) has a low TSlevel, a recommendation can be made to treat or to not treat the subjectwith adjuvant 5-FU-based chemotherapy.

The status of Topol expression levels in a sample from a subject, e.g.,a colon cancer patient, can be determined by, e.g., IHC, and can be usedto determine whether to treat the subject with irinotecan.Progression-free survival (PFS) was not improved in patients with lowTopol by the addition of irinotecan, but patients with moderate/highTopol benefited from the addition of irinotecan (Braun M S et al. (2008)J Clin. Oncol. 26:2690-2698). If a sample from a subject has low Topolexpression, then a recommendation can be made to not treat the subjectwith irinotecan. If a sample from a subject has moderate to high Topolexpression, then a recommendation can be made to treat the subject withirinotecan.

The status of Kras in a sample from a colon cancer patient can bedetermined by, e.g., sequencing, and bevacizumab (Avastin) can berecommended as a drug treatment option based on the status of Kras.

The status of TOPO1 in a sample from a colon cancer patient can bedetermined by, e.g., immunohistochemistry (IHC) and/or sequencing, andcan be used to determine whether to recommend 5-FU or capecitabine(Xeloda) as a drug treatment option. For example, if TOPO1 expressioncan be detected by IHC or if TOPO1 has a certain sequence, thencapecitabine (Xeloda), or fluorouracil (5-FU) with or without irinotecan(Camptosar), can be recommended as a drug treatment option.

Combinations of markers/targets, drug/therapeutics, and cancers can befound in FIG. 6.

D. Drugs with Inhibitors of DNA Synthesis

Other cancer drug therapies that can be recommended to a subjectinclude, for example, FOLFOX. FOLFOX is a chemotherapy regimen that canbe used to treat colorectal cancer. The FOLFOX regimen includes folinicacid (leucovorin), fluorouracil (5-FU), and oxaliplatin (Eloxatin). 5-FUcan act as an inhibitor of thymidylate synthase, which can blocksynthesis of thymidine, which can affect DNA replication. Capecitabine(Xeloda) is a prodrug that can be converted enzymatically to 5-FU.

Capecitabine (Xeloda) can be used to treat metastatic breast cancer andcolorectal cancer. Irinotecan (Camptosar) is an inhibitor oftopoisomerase 1 that can be used for treatment of colon cancer. FOLFIRIis a chemotherapy regimen that can be used to treat colorectal cancer.The FOLFIRI regimen includes folinic acid (leucovorin), fluorouracil(5-FU), irinotecan (Camptosar). CapeOx is a chemotherapy regimen thatincludes capecitabine (Xeloda) and oxaliplatin (Eloxatin).

E. Patient Metabolism

The stratifying of cancer drug treatments can take into account the roleof genes that affect drug absorption, distribution, metabolism, andexcretion (the pharmacokinetics and pharmacodynamics of the drugtreatment). The stratifying of drug treatments can take into accountwhether the subject or patient is hypermetabolic. The stratifying ofdrug treatments can take into account the cytochrome P450 (CYP450)status of said subject or patient. The CYP450 status of a subject or apatient can be determined, for example, from assessments made bygenotyping genes encoding CYP450 (e.g., CYP2D6, CYP2C19, and CYP2C9).For example, the CYP450 status can be assessed using the Roche®AmpliChip CYP450 Test, which tests for gene variations in CYP2D6 andCYP2C19. The stratifying of one or more drug treatment options can bebased on tests for defects in the enzyme thiopurine methyltransferase(TMPT), which can prevent metabolism of the anti-cancer drug6-mercaptopurine (6MP). Results from Affymetrix® Drug MetabolizingEnzymes and Transporters (DMET)—Early Access Solution, The Human1MBeadChip from Illumina®, Human1M-Duo DNA Analysis BeadChip fromIllumina®, and HumanExon510S-Duo DNA Analysis BeadChip from Illumina®,can be used to stratify the cancer drug treatment options.

F. Sources of Drug Treatment Options

The list of drug treatments to be stratified can be compiled frominformation sources available to those skilled in the art. The drugs canbe described in scientific literature, and the drugs can be those usedin unpublished clinical trials (clinicaltrials.gov/). The drugs can belisted in the Centers for Medicare and Medicaid Services (CMS)anti-cancer treatment compendia for determining which drugs may becovered under Medicare Part B to treat cancer patients, including theNational Comprehensive Cancer Network (NCCN) Drugs and BiologicsCompendium™ Thomson Micromedex DrugDex®, and Elsevier Gold Standard'sClinical Pharmacology compendium, and American Hospital FormularyService—Drug Information Compendium. The drugs can be those listed for aspecific cancer on the NCCN Clinical Practice Guidelines in Oncology™website or in the American Society of Clinical Oncology (ASCO) clinicalpractice guidelines.

G. Types of Cancers

The NCCN Clinical Practice Guidelines in Oncology™ website providesclinical practice guidelines for treating a variety of cancers. Theseclinical practice guidelines can be downloaded. The guidelines provideinformation regarding care options that take into account risks andbenefits associated with a procedure.

The condition for which one or more drug treatment options can bestratified and/or annotated can include a cancer listed on the NCCNwebsite. The conditions or cancers can include, for example, acutemyeloid leukemia; bladder cancer, including upper tract tumors andurothelial carcinoma of the prostate; bone cancer, includingchondrosarcoma, Ewing's sarcoma, and osteosarcoma; breast cancer,including noninvasive, invasive, phyllodes tumor, Paget's disease, andbreast cancer during pregnancy; central nervous system cancers, adultlow-grade infiltrative supratentorial astrocytoma/oligodendroglioma,adult intracranial ependymoma, anaplastic astrocytoma/anaplasticoligodendroglioma/glioblastoma multiforme, limited (1-3) metastaticlesions, multiple (>3) metastatic lesions, carcinomatous lymphomatousmeningitis, nonimmunosuppressed primary CNS lymphoma, and metastaticspine tumors; cervical cancer; chronic myelogenous leukemia (CML); coloncancer, rectal cancer, anal carcinoma; esophageal cancer; gastric(stomach) cancer; head and neck cancers, including ethmoid sinus tumors,maxillary sinus tumors, salivary gland tumors, cancer of the lip, cancerof the oral cavity, cancer of the oropharynx, cancer of the hypopharynx,occult primary, cancer of the glottic larynx, cancer of the supraglotticlarynx, cancer of the nasopharynx, and advanced head and neck cancer;hepatobiliary cancers, including hepatocellular carcinoma, gallbladdercancer, intrahepatic cholangiocarcinoma, and extrahepaticcholangiocarcinoma; Hodgkin disease/lymphoma; kidney cancer; melanoma;multiple myeloma, systemic light chain amyloidosis, Waldenstrom'smacroglobulinemia; myelodysplastic syndromes; neuroendocrine tumors,including multiple endocrine neoplasia, type 1, multiple endocrineneoplasia, type 2, carcinoid tumors, islet cell tumors,pheochromocytoma, poorly differentiated/small cell/atypical lungcarcinoids; Non-Hodgkin's Lymphomas, including chronic lymphocyticleukemia/small lymphocytic lymphoma, follicular lymphoma, marginal zonelymphoma, mantle cell lymphoma, diffuse large B-Cell lymphoma, Burkitt'slymphoma, lymphoblastic lymphoma, AIDS-Related B-Cell lymphoma,peripheral T-Cell lymphoma, and mycosis fungoides/Sezary Syndrome;non-melanoma skin cancers, including basal and squamous cell skincancers, dermatofibrosarcoma protuberans, Merkel cell carcinoma;non-small cell lung cancer (NSCLC), including thymic malignancies;occult primary; ovarian cancer, including epithelial ovarian cancer,borderline epithelial ovarian cancer (Low Malignant Potential), and lesscommon ovarian histologies; pancreatic adenocarcinoma; prostate cancer;small cell lung cancer and lung neuroendocrine tumors; soft tissuesarcoma, including soft-tissue extremity, retroperitoneal,intra-abdominal sarcoma, and desmoid; testicular cancer; thymicmalignancies, including thyroid carcinoma, nodule evaluation, papillarycarcinoma, follicular carcinoma, Hürthle cell neoplasm, medullarycarcinoma, and anaplastic carcinoma; uterine neoplasms, includingendometrial cancer and uterine sarcoma.

Medical information regarding cancers can be found at websites, forexample, www.cancer.gov, www.nexcura.cancer.com, www.asco.org, andnccn.org/professionals/physician.

H. How Stratification is Indicated on a Report

The stratifying of drug treatment options can be indicated by markingson a form downloaded from a website, for example, the NCCN ClinicalPractice Guidelines in Oncology™ or in the American Society of ClinicalOncology (ASCO) clinical practice guidelines, or on a printed report.For example, color coding of drug treatment options can be used toindicate stratification of drug treatment options on informationdownloaded or printed, for example, from the NCCN Clinical PracticeGuidelines in Oncology™ website. The drug treatment options that can bestratified include those found in the American Society of ClinicalOncology (ASCO) clinical practice guidelines. The drug treatment optionscan be stratified such that drug treatment options most likely to beefficacious (first class; recommend the subject be treated with thedrug), based on the status of one of more molecular markers of thesample or tumor, are highlighted or written in green or a shade ofgreen; drugs that may have some efficacy are highlighted or written inyellow or a shade of yellow (second class; recommend the subject betreated with the drug with some caution or caveat), and drugs for whichno information is available or for which the scientific literatureindicates the drug will not be efficacious or will be toxic in light ofthe status of the subject's one or more molecular markers arehighlighted or written in red or a shade of red (third class; recommendthe subject not be treated with the drug). FIGS. 2A-2C illustrate anexample in which (FIG. 2A) a health care provider accesses a report on acomputer based on (FIG. 2B) the NCCN Clinical Practice Guidelines inOncology related to colon cancer, and (FIG. 2C) drugs treatment optionsare classified (stratified) by colors. FIG. 8 illustrates a printed hardcopy example of a report in which drug, target (molecular marker), andstatus of a target (molecular marker) are indicated. Other identifiers,such as number ranking, separate groups, asterisks, etc., can be used toindicate stratification of the drug treatment options for a condition.

The report can contain other information, including, e.g., type of assayor technique used for determining the status of a molecular marker,different type of molecular marker status (e.g., wild-type or mutantsequence; e.g., DNA, RNA, or protein sequence; high expression or lowexpression levels, e.g., mRNA expression or protein expression; etc.).The report can contain information on a drug dosing recommendation basedon the status of one or molecular markers.

The report with the stratified treatment options can be provided by thepersonalized medicine business to a health care professional (e.g.,oncologist) and/or the subject for whom the report is prepared. Thereport can be made available online or be delivered in hardcopy form bya delivery service. An alert, such as an email, text message, phonecall, facsimile, etc. can be sent to a subject or health care providerindicating that the report is available. A fee can be charged by thepersonalized medicine business in exchange for preparing and/or sendingthe report.

I. Validation of Tumor Markers

The methods of the provided invention can be used to validate molecularmarkers. A molecular marker to be validated for a particular cancer canbe selected by analyzing the literature. Stored samples from subjectswhose treatment outcome is known (retrospective sample) can be used tovalidate a molecular marker of a condition, e.g., a cancer. Validating atumor marker can include determining a DNA sequence variation andcorrelating the variation to RNA expression. Hundreds of markers can bevalidated in parallel. Quantitative cellular heterogeneity can be usedas a measure of survival. A validation study can be a FFPE retrospectivevalidation study. A validation study can be a prospective validationstudy. A validation study can be a multi-marker retrospectivecorrelation study.

V. Annotating Cancer Drug Treatment Options

A. Annotating with Information Regarding Experimental Drugs andFDA-Approved Drugs for Off-Label Use

The methods of the provided invention can include obtaining a sample,determining the status of one or more molecular markers, stratifying oneor more drug treatment options based on the status of the one or moremolecular markers, and annotating drug treatment options in a reportbased on the status of the one or more molecular markers. The annotatedinformation can be used by a health care provider to select other drugtreatment options and/or provide information about drug treatmentoptions to an insurance company. The method can include annotating thedrug treatment options for a condition in, for example, the NCCNClinical Practice Guidelines in Oncology™ or the American Society ofClinical Oncology (ASCO) clinical practice guidelines.

The drug treatment options that are stratified in a report can beannotated in the report by listing additional drug treatment options. Anadditional drug treatment can be an FDA-approved drug for an off-labeluse. A provision in the 1993 Omnibus Budget Reconciliation Act (OBRA)requires Medicare to cover off-label uses of anticancer drugs that areincluded in standard medical compendia. The drugs used for annotatinglists can be found in CMS approved compendia, including the NationalComprehensive Cancer Network (NCCN) Drugs and Biologics Compendium™,Thomson Micromedex DrugDex®, Elsevier Gold Standard's ClinicalPharmacology compendium, and American Hospital Formulary Service—DrugInformation Compendium®.

The drug treatment options can be annotated by listing an experimentaldrug that may be useful in treating a cancer with one or more molecularmarkers of a particular status. The experimental drug can be a drug forwhich in vitro data, in vivo data, animal model data, pre-clinical trialdata, or clinical-trial data are available. The data can be published inpeer-reviewed medical literature found in journals listed in the CMSMedicare Benefit Policy Manual, including, for example, American Journalof Medicine, Annals of Internal Medicine, Annals of Oncology, Annals ofSurgical Oncology, Biology of Blood and Marrow Transplantation, Blood,Bone Marrow Transplantation, British Journal of Cancer, British Journalof Hematology, British Medical Journal, Cancer, Clinical CancerResearch, Drugs, European Journal of Cancer (formerly the EuropeanJournal of Cancer and Clinical Oncology), Gynecologic Oncology,International Journal of Radiation, Oncology, Biology, and Physics, TheJournal of the American Medical Association, Journal of ClinicalOncology, Journal of the National Cancer Institute, Journal of theNational Comprehensive Cancer Network (NCCN), Journal of Urology,Lancet, Lancet Oncology, Leukemia, The New England Journal of Medicine,and Radiation Oncology.

B. Annotating with Scientific Information about the Drugs

The drug treatment options can be annotated by providing a link on anelectronic based report connecting a listed drug to scientificinformation regarding the drug. For example, a link can be provided toinformation regarding a clinical trial for a drug (clinicaltrials.gov).If the report is provided via a computer or computer website, the linkcan be a footnote, a hyperlink to a website, a pop-up box, or a fly-overbox with information, etc. The annotated information can becomeavailable when a computer user clicks on a condition or a treatmentoption on the downloaded and annotated Clinical Practice Guidelines inOncology™ (FIG. 3). The report and the annotated information can beprovided on a printed form, and the annotations can be, for example, afootnote to a reference.

The information for annotating one or more drug treatment options in areport can be provided by a commercial entity that stores scientificinformation, for example, Ingenuity® Systems. A health care provider cantreat a subject, such as a cancer patient, with an experimental druglisted in the annotated information, and the health care provider canaccess the annotated drug treatment option, retrieve the scientificinformation (e.g., print a medical journal article) and submit it (e.g.,a printed journal article) to an insurance company along with a requestfor reimbursement for providing the drug treatment. Physicians can useany of a variety of Diagnosis-related group (DRG) codes to enablereimbursement.

A drug treatment option in a report can also be annotated withinformation regarding other molecular components in a pathway that adrug affects (e.g., information on a drug that targets a kinasedownstream of a cell-surface receptor that is a drug target). The drugtreatment option can be annotated with information on drugs that targetone or more other molecular pathway components. The identificationand/or annotation of information related to pathways can be outsourcedor subcontracted to another company, for example Ingenuity®.

The annotated information can be, for example, a drug name (e.g., an FDAapproved drug for off-label use; a drug found in a CMS approvedcompendium, and/or a drug described in a scientific (medical) journalarticle), scientific information concerning one or more drug treatmentoptions, one or more links to scientific information regarding one ormore drugs, clinical trial information regarding one or more drugs(e.g., information from clinicaltrials.gov/), one or more links tocitations for scientific information regarding drugs, etc.

The annotated information can be inserted into any location in a report.Annotated information can be inserted in multiple locations on a report.Annotated information can be inserted in a report near a section onstratified drug treatment options. Annotated information can be insertedinto a report on a separate page from stratified drug treatment options.A report that does not contain stratified drug treatment options can beannotated with information.

VI. Screening Drugs Using Xenograft Models and In Vitro Cultures

The provided methods can also include means for investigating theeffects of drugs on sample (e.g. tumor cells) isolated from a subject(e.g. cancer patient). An in vitro culture using a tumor from a cancerpatient can be established using techniques known to those skilled inthe art.

The provided method can also include establishing a xenograft modelusing said sample. The sample can be a tumor biopsy from a humansubject. Cells from tumor biopsy can be transplanted to a species thatincludes, for example, a pig, mouse, severe combined immunodeficiency(SCID) mouse, rat, nude rat, etc.

The provided method can also include high-throughput screening of FDAapproved off-label drugs or experimental drugs using said in vitroculture and/or xenograft model.

The provided method can also include monitoring tumor antigen forrecurrence detection.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES Prophetic Example 1: Stratifying Colon Cancer Drug TreatmentOptions

A colon cancer patient visits a physician, and the physician performs atumor biopsy. The physician arranges to submit the tumor biopsy to apersonalized medicine business for characterization of molecularmarkers. In the meantime, the physician prescribes a first line drugtreatment for the cancer patient selected from those disclosed in theNCCN Clinical Practice Guidelines in Oncology™ guidelines for ColonCancer.

The tumor biopsy is obtained by the personalized medicine business, andprimary tumor cells are separated from non-tumor cells by flow-sorting.The isolated primary tumor cells are tested for the status of one ormore molecular marker(s) by comparative genomic hydridization (CGH), DNAsequencing, and high density expression. The status of the one or moremolecular markers is used to analyze scientific publications forliterature related to responses of tumors with molecular markers of asimilar status to drug treatment options. The analysis is completed byone or more individuals with knowledge or expertise in colon cancertreatment employed by the personalized medicine business or outsourcedor subcontracted to another company. The treatment options listed in theNCCN Clinical Practice Guidelines in Oncology™ guidelines for ColonCancer are evaluated in light of the status of the one or more molecularmarkers identified in the tumor cells and in light of availablescientific information regarding those markers and colon or other cancerdrug treatment options. An individual or teams ranks or classifies thedrug treatment options based on predicted efficacy given the status ofthe molecular markers in the cancer patient's sample. Those drugtreatment options predicted to have the highest efficacy are highlightedin green, those predicted to have lower efficacy are highlighted inyellow, and those for which a marker indicates a drug will not beefficacious or will be toxic are indicated in red.

In addition, scientific literature and clinical trial information arereviewed for other drug treatment options not listed in the NCCNClinical Practice Guidelines in Oncology™ guidelines for Colon Cancer.Based on these information sources, a report containing the NCCNClinical Practice Guidelines in Oncology™ guidelines for Colon Cancer isannotated by listing other drug treatment options that includeFDA-approved drugs for an off-label use and/or experimental drugs. Thedownloaded version of the guidelines lists the annotated drug, and alink is provided from each listed drug treatment option to scientificinformation related to the use of that drug.

The stratified drugs treatment options and annotated drug treatmentoptions are shared with the patient and/or the physician in the form ofan electronic or hard copy report. The physician and/or patient make asecond line treatment decision to use an experimental drug based on themolecular marker data. The health care provide can access the modifiedNCCN Clinical Practice Guidelines in Oncology™ guidelines for ColonCancer on a computer, point to the annotated drug, which brings up apop-box with a link to a journal article concerning the experimentaldrug. The health care provider can submit the journal article to aninsurance provider to seek reimbursement for the treatment.

Prophetic Example 2: Xenograft and In Vitro Cultures

A colon cancer patient visits a physician, and the physician performs atumor biopsy. The physician submits the biopsy to a personalizedmedicine business for molecular characterization. In addition, an invitro culture and nude rat xenograft model are established. A series ofexperimental drugs are identified as described in Prophetic Example 1that may be helpful for the patient. While the patient is in a firstline drug therapy, a high-throughput screen is performed with theexperimental drugs and the in vitro culture and xenograft models.Results of the high-throughput screen are reported to the patient andthe physician, who use the information in deciding on a second-linetherapy if the first line therapy fails or an adjunct in the end stageadministration if appropriate.

Prophetic Example 3: Annotating Treatment Options on the NCCN Website

The scientific literature lists a treatment X for cancer A and cancer B.Treatment Y is useful in treating cancer B, but the affect of treatmentY on cancer A, however, is not known or studied. A report with the NCCNClinical Practice Guidelines in Oncology™ on a website is annotated withinformation describing the relationship between treatment Y and cancer Band treatment X and cancers A and B, and a note is added that treatmentY may be effective for off-label use in treating cancer A.

Prophetic Example 4: Physician Use of NCCN Website with StratifiedTreatment Options

A physician (FIG. 2A) accesses a NCCN Clinical Practice Guidelines inOncology™ file on Colon Cancer (FIG. 2B) in which a patient's treatmentoptions have been stratified based on the status of the patient'smolecular markers (FIG. 2C). The highest ranked drug treatment optionfor a patient with marker X and is marked in green (Drug A). The nextranked drug is marked in yellow (Drug B). The drug marked in red (DrugC) does not display efficacy based on the status of the molecular markerX.

Prophetic Example 5: Physician Use of NCCN Website with AnnotatedTreatment Options

A physician accesses a NCCN Clinical Practice Guidelines in Oncology™file on Colon Cancer that has been annotated with an additional drugtreatment option D (FIG. 3; 300). Option D is an experimental drug withpublished data in vitro on treating a tumor with a marker whose statusis shared with the physician's patient. The physician treats the patientwith drug D (302). The physician follows the link on the page to amedical journal article on the use of drug D in the in vitro study (304)and submits the article to an insurance company with a request forreimbursement (306).

FIG. 8 depicts a report with annotated treatment options. The markers(targets), status of the markers (targets), and drugs are listed.Annotated information appears in multiple places on the report.

Prophetic Example 6: Stratification and Annotation of a CombinationRegimen Drug Treatment Option

An oncologist submits an order to a personalized medicine business onbehalf of a colon cancer patient. The oncologist submits a tumor biopsyfrom the colon cancer patient to the personalized medicine business, andthe personalized medicine business identifies molecular markers in thesample. FOLFOX is a chemotherapy regimen that can be used to treatcolorectal cancer. The FOLFOX treatment regimen includes the drugsfolinic acid (leucovorin), fluorouracil (5-FU), and oxaliplatin(Eloxatin). The molecular marker information in the sample from a coloncancer patient indicates that two of the drugs in FOLFOX will beefficacious, while the molecular marker information indicates that oneof the drugs will not. The personalized medicine business highlights thedrug in yellow in the list of drug treatment options, and annotates thereport with scientific information. A computer downloadable report isprepared by the personalized medicine business and transmitted to thesubject and the subject's oncologist with the FOLFOX drug listinghighlighted in yellow. When the subject or the oncologist click on theFOLFOX drug listing, a pop-up box appears that indicates the two drugsin the FOLFOX regimen that are likely to be efficacious and the one drugthat is not likely to be efficacious in treating the subject.

Prophetic Example 7: Platinum-Based Drug Treatment Options

Platinum-based drug treatment options include cisplatin, caroplatin,picoplatin, satraplatin, oxaliplatin. The efficacy or resistance of someplatinum-based therapies can be correlated with certain expressionprofiles. A platinum-based drug is listed as a drug treatment option ina report and is annotated to indicate which expression profiles arecorrelated with efficacy or resistance.

Prophetic Example 8: An Aspect of a Method of the Provided Invention

A health care provider performs a diagnostic biopsy of a primary tumorin a subject. A therapy is begun, and the tumor is tested for the statusof one or more molecular markers (402). An order is placed to thepersonalized medicine business (404). A physician places the order, orassists the subject (e.g., cancer patient) in placing the order. Theorder can contain clinical information and molecular informationregarding the subject and the tumor of the subject at the time the orderis placed (404). The order can be placed on a website (404). Theclinical and/or molecular information is transmitted to the personalizedmedicine business via one or more paper documents, emails, orfacsimiles. A customer service representative employed by thepersonalized medicine business processes the order and a research nurseprocesses the clinical and molecular information (404).

One or more kits are sent by the personalized medicine business beforethe second biopsy (406). The patient and an oncologist are notified thata kit is sent to a pathologist, and the patient and the oncologist arenotified that a kit is sent to a surgeon (406). The kit sent to thepathologist contains instructions for preparing a diagnostic paraffinprimary biopsy, an envelope for sending the sample to the personalizedmedicine business, and identifier information (406). The kit sent to thesurgeon contains RNAlater® and instructions for freezing the sample(406). A kit is presented to the pathologist after the surgery (406). Alogistics and operations team employed by the personalized medicinebusiness sends the kits (406), and a fee is charged for sending the kits(406).

A biopsy of the sample is performed by a surgeon and the sample isfrozen (408). The frozen sample is sent by FedEx in a box with dry ice(406). The frozen tissue is sent in a tube, and a blood sample from thepatient is sent to the personalized medicine business for analyzingmetabolism genes (406). Specific directions are provided for sending thesample (406). Parafilm embedded, frozen, and/or fine needle biopsies aresent to the personalized medicine business (406).

A pathologist reviews or blocks the samples (410). Cancer cells arepurified from a sample that is sent (412). The cancer cells are purifiedby flow sorting (using markers and/or ploidy) or by laser capturemicroscopy (412). Two full-time employees (FTE) of the personalizedmedicine business are available to purify the cancer cells. A fee ischarged in exchange for purifying the cancer cells.

Cells, e.g., circulating tumor cells, can be isolated from a sample,e.g., blood, using technology from, e.g., CELLective Dx Corporation.

The purified cancer cells are analyzed by a variety of assays by thepersonalized medicine business. The personalized medicine businessperforms targeted assays dependent on the tumor type (414). A logisticsand operations employee and two laboratory technicians perform theseservices (414). A fee is charged for the services (414). Some of thetargeted assays are outsourced (414).

The personalized medicine business performs whole genome analysis assays(416). These assays include comparative genomic hybridization (CGH),high density expression, analysis for small nucleotide polymorphisms(SNPs), proteins assays, and sequencing (416). Up to six laboratorytechnicians perform these tests, and a fee is charged in exchange forperforming these tests.

Molecular tests are performed to analyze drug metabolism genes (418).These tests include the Affymetrix® Drug Metabolizing Enzymes andTransporters (DMET)—Early Access Solution, Human1M BeadChip fromIllumina®, Human1M-Duo DNA Analysis BeadChip from Illumina®,HumanExon510S-Duo DNA Analysis BeadChip from Illumina®, and the Roche®AmpliChip CYP450 Test (418). Laboratory technicians employed by thepersonalized medicine business perform the tests. The personalizedmedicine business charges a fee in exchange for performing the tests.

Other in vitro and in vivo assays are outsourced by the personalizedmedicine business to another company (420).

The information from the targeted assays, whole genome assays, drugmetabolism tests, and/or in vitro and in vivo assays are provided toIngenuity®. Ingenuity® as well as bioinformatics employees of thepersonalized medicine business identify scientific information relatedto cancer drugs and the status of the molecular markers in the patient'ssample(s) (422). A molecular pathologist and research nurse annotate thedrug information with links to appropriate scientific literature (424).A report is sent to an oncologist (426).

Prophetic Example 9: Drug Treatment Recommendations

A cancer patient contacts a personalized medicine business to obtain areport regarding personalized drug treatment options based on the statusof molecular markers in his tumor. The cancer patient submits a sampleto the personalized medicine business. DNA is extracted from the sampleand is subjected to massively parallel DNA sequencing. The sample isalso subjected to qPCR. The sequence of Kras is determined and EGFR copynumber is determined. The Kras sequence is determined to be wild-type,and EGFR copy number is determined to be higher than normal. A report isprepared in which cetuximab (Erbitux) and panitumumab (Vectibix) arerecommended for the colon cancer patient. The drug treatment optionnames are highlighted in green on the report. Each drug treatment optionis annotated with a number for a footnote, which list references thatsupport the use of cetuximab (Erbitux) and panitumumab (Vectibix) forcancer patients with tumors with wild-type Kras and increased EGFR copynumber.

What is claimed is:
 1. A method of determining a status of more than onegene from a formalin-fixed paraffin embedded biopsy from a subject, saidmethod comprising: (a) obtaining a formalin-fixed paraffin embedded(FFPE) biopsy, wherein said FFPE biopsy comprises nucleic acid; and (b)determining a status of more than one gene in said nucleic acid, whereinsaid determining comprises de novo DNA sequencing of said nucleic acid.2. The method of claim 1, wherein said FFPE biopsy comprises a tumorcell.
 3. The method of claim 1, wherein said nucleic acid comprises DNA.4. The method of claim 1, wherein said nucleic acid comprises genomicDNA.
 5. The method of claim 1, further comprising amplifying saidnucleic acid from said FFPE biopsy.
 6. The method of claim 1, whereinsaid determining said status of said more than one gene comprisesdetermining a presence or absence of a point mutation, a singlenucleotide polymorphism, copy number variation, substitution, insertion,deletion, rearrangement, de novo mutation, nonsense mutation, missensemutation, silent mutation, frameshift mutation, amplification,chromosomal translocation, interstitial deletion, chromosomal inversion,loss of heterozygosity, loss of function mutation, gain of functionmutation, dominant negative mutation, or lethal mutation.
 7. The methodof claim 1, wherein said determining said status of said more than onegene comprises determining a presence or absence of a substitution,insertion, deletion, rearrangement, or amplification.
 8. The method ofclaim 1, wherein said determining said status of said more than one genecomprises determining a presence or absence of a copy number variation.9. The method of claim 1, wherein said determining said status of saidmore than one gene comprises determining a presence or absence of apoint mutation.
 10. The method of claim 1, wherein said determining saidstatus of said more than one gene comprises determining a presence orabsence of a single nucleotide polymorphism.
 11. The method of claim 1,wherein said determining said status of said more than one genecomprises determining a presence or absence of an amplification.
 12. Themethod of claim 1, wherein said de novo DNA sequencing comprisesattaching said nucleic acid to a solid surface.
 13. The method of claim1, wherein said de novo DNA sequencing comprises bridge amplification.14. The method of claim 1, wherein said de novo DNA sequencing comprisesuse of reversibly terminating nucleotides.
 15. The method of claim 1,further comprising performing digital polymerase chain reaction (PCR) onsaid FFPE biopsy.
 16. The method of claim 1, wherein said more than onegene comprises KIT, BRAF, Hsp90AA1, EGFR, KRAS, ESR1, TOPOI, PTEN,PIK3CA, NFE2L2, and ERBB2.
 17. The method of claim 1, wherein said morethan one gene comprises KIT, BRAF, EGFR, ERBB2, ESR1, KRAS, and PIK3CA.18. The method of claim 1, wherein said more than one gene comprisesKRAS, EGFR, BRAF, ERBB2, and KIT.
 19. The method of claim 1, whereinsaid more than one gene comprises KRAS, EGFR, BRAF, and KIT.
 20. Themethod of claim 1, wherein said more than one gene comprises KRAS. 21.The method of claim 1, wherein said more than one gene comprises PIK3CA.22. The method of claim 1, wherein said FFPE biopsy comprises a tumorcell from a colon cancer, a bone cancer, a breast cancer, a centralnervous system cancer, a gastric cancer, a cervical cancer, a bloodcancer, an esophageal cancer, a head and neck cancer, a kidney cancer, askin cancer, a lung cancer, or a carcinoma.
 23. The method of claim 1,wherein said FFPE biopsy comprises a tumor cell from a colon cancer. 24.The method of claim 1, wherein said FFPE biopsy comprises a tumor cellfrom blood.
 25. The method of claim 1, wherein said determining saidstatus of more than one gene comprises hybridizing a probe nucleic acidto nucleic acids corresponding to said more than one gene from a tumorcell.
 26. The method of claim 1, wherein said de novo DNA sequencingcomprises use of a chemical-sensitive field effect transistor (chemFET)array.
 27. The method of claim 1, wherein said de novo DNA sequencingcomprises using a laser to excite a fluorophore.
 28. The method of claim1, further comprising ligating an adaptor to said nucleic acid.
 29. Themethod of claim 1, wherein said subject is a human.
 30. The method ofclaim 1, wherein said obtaining comprises sending said sample through amail or delivery service.
 31. The method of claim 1, further comprisingobtaining additional samples after obtaining an initial sample from saidsubject, wherein said additional samples comprise FFPE biopsy comprisingDNA from a tumor cell.
 32. The method of claim 1, wherein said de novoDNA sequencing occurs in a Clinical Laboratory Improvement Amendment(CLIA) certified laboratory.